Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 achieves global cardiac gene transfer in canines

PubMed Central

Bish, Lawrence T.; Sleeper, Meg M.; Brainard, Benjamin; Cole, Stephen; Russell, Nicholas; Withnall, Elanor; Arndt, Jason; Reynolds, Caryn; Davison, Ellen; Sanmiguel, Julio; Wu, Di; Gao, Guangping; Wilson, James M.; Sweeney, H. Lee

2011-01-01

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus or plasmid DNA. While single stranded adeno-associated virus vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented by using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated virus vectors to the canine myocardium. Four vectors were evaluated—single stranded adeno-associated virus 9, self-complementary adeno-associated virus 9, self-complementary adeno-associated virus 8, self-complementary adeno-associated virus 6—so that comparison could be made between single stranded and self complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that self-complementary adeno-associated virus is superior to single stranded adeno-associated virus and that adeno-associated virus 6 is superior to other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15 to 4000 times more abundant in the heart than in any other organ for self-complementary adeno-associated virus 6. Percutaneous transendocardial injection of self-complementary adeno-associated virus 6 is a safe, effective method for achieving efficient cardiac gene transfer. PMID:18813281

Nerve Growth Factor Gene Therapy Using Adeno-Associated Viral Vectors Prevents Cardiomyopathy in Type 1 Diabetic Mice

PubMed Central

Meloni, Marco; Descamps, Betty; Caporali, Andrea; Zentilin, Lorena; Floris, Ilaria; Giacca, Mauro; Emanueli, Costanza

2012-01-01

Diabetes is a cause of cardiac dysfunction, reduced myocardial perfusion, and ultimately heart failure. Nerve growth factor (NGF) exerts protective effects on the cardiovascular system. This study investigated whether NGF gene transfer can prevent diabetic cardiomyopathy in mice. We worked with mice with streptozotocin-induced type 1 diabetes and with nondiabetic control mice. After having established that diabetes reduces cardiac NGF mRNA expression, we tested NGF gene therapies with adeno-associated viral vectors (AAVs) for the capacity to protect the diabetic mouse heart. To this aim, after 2 weeks of diabetes, cardiac expression of human NGF or β-Gal (control) genes was induced by either intramyocardial injection of AAV serotype 2 (AAV2) or systemic delivery of AAV serotype 9 (AAV9). Nondiabetic mice were given AAV2–β-Gal or AAV9–β-Gal. We found that the diabetic mice receiving NGF gene transfer via either AAV2 or AAV9 were spared the progressive deterioration of cardiac function and left ventricular chamber dilatation observed in β-Gal–injected diabetic mice. Moreover, they were additionally protected from myocardial microvascular rarefaction, hypoperfusion, increased deposition of interstitial fibrosis, and increased apoptosis of endothelial cells and cardiomyocytes, which afflicted the β-Gal–injected diabetic control mice. Our data suggest therapeutic potential of NGF for the prevention of cardiomyopathy in diabetic subjects. PMID:22187379

Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.

PubMed

Wang, Zhong; Zhu, Tong; Qiao, Chunping; Zhou, Liqiao; Wang, Bing; Zhang, Jian; Chen, Chunlian; Li, Juan; Xiao, Xiao

2005-03-01

Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventions to enforce broad vector distribution. We compared the efficiency of multiple adeno-associated virus (AAV) vectors after a single injection via intraperitoneal or intravenous routes without additional intervention. We show that AAV8 is the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles of mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier. Gene expression persisted in muscle and heart, but diminished in tissues undergoing rapid cell division, such as neonatal liver. This technology should prove useful for muscle-directed systemic gene therapy.

Molecular Analysis of Vector Genome Structures After Liver Transduction by Conventional and Self-Complementary Adeno-Associated Viral Serotype Vectors in Murine and Nonhuman Primate Models

PubMed Central

Sun, Xun; Lu, You; Bish, Lawrence T.; Calcedo, Roberto; Wilson, James M.

2010-01-01

Abstract Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage ϕ29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations. PMID:20113166

Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert

2012-09-17

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pHmore » 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.« less

Adeno-Associated Viral Vector Serotype DJ-Mediated Overexpression of N171-82Q-Mutant Huntingtin in the Striatum of Juvenile Mice Is a New Model for Huntington's Disease.

PubMed

Jang, Minhee; Lee, Seung Eun; Cho, Ik-Hyun

2018-01-01

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. HD is caused by an expansion of CAG repeats in the huntingtin ( HTT ) gene in various areas of the brain including striatum. There are few suitable animal models to study the pathogenesis of HD and validate therapeutic strategies. Recombinant adeno-associated viral (AAV) vectors successfully transfer foreign genes to the brain of adult mammalians. In this article, we report a novel mouse model of HD generated by bilateral intrastriatal injection of AAV vector serotype DJ (AAV-DJ) containing N171-82Q mutant HTT (82Q) and N171-18Q wild type HTT (18Q; sham). The AAV-DJ-82Q model displayed motor dysfunctions in pole and rotarod tests beginning 4 weeks after viral infection in juvenile mice (8 weeks after birth). They showed behaviors reflecting neurodegeneration. They also showed increased apoptosis, robust glial activation and upregulated representative inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), mediators (cyclooxygenase-2 and inducible nitric oxide synthase) and signaling pathways (nuclear factor kappa B and signal transducer and activator of transcription 3 (STAT3)) in the striatum at 10 weeks after viral infection (14 weeks after birth) via successful transfection of mutant HTT into neurons, microglia, and astrocytes in the striatum. However, little evidence of any of these events was found in mice infected with the AAV-DJ-18Q expressing construct. Intrastriatal injection of AAV-DJ-82Q might be useful as a novel in vivo model to investigate the biology of truncated N-terminal fragment (N171) in the striatum and to explore the efficacy of therapeutic strategies for HD.

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

PubMed Central

Janovitz, Tyler; Klein, Isaac A.; Oliveira, Thiago; Mukherjee, Piali; Nussenzweig, Michel C.; Sadelain, Michel

2013-01-01

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics. PMID:23720718

Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

PubMed

Earley, Lauriel F; Powers, John M; Adachi, Kei; Baumgart, Joshua T; Meyer, Nancy L; Xie, Qing; Chapman, Michael S; Nakai, Hiroyuki

2017-02-01

Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly. Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice

PubMed Central

Mak, Kai Y; Chin, Ruth; Cunningham, Sharon C; Habib, Miriam R; Torresi, Joseph; Sharland, Alexandra F; Alexander, Ian E; Angus, Peter W; Herath, Chandana B

2015-01-01

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1–7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1–7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects. PMID:25997428

Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

ERIC Educational Resources Information Center

Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

2010-01-01

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

Adeno-Associated Viral Vector Serotype DJ-Mediated Overexpression of N171-82Q-Mutant Huntingtin in the Striatum of Juvenile Mice Is a New Model for Huntington’s Disease

PubMed Central

Jang, Minhee; Lee, Seung Eun; Cho, Ik-Hyun

2018-01-01

Huntington’s disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. HD is caused by an expansion of CAG repeats in the huntingtin (HTT) gene in various areas of the brain including striatum. There are few suitable animal models to study the pathogenesis of HD and validate therapeutic strategies. Recombinant adeno-associated viral (AAV) vectors successfully transfer foreign genes to the brain of adult mammalians. In this article, we report a novel mouse model of HD generated by bilateral intrastriatal injection of AAV vector serotype DJ (AAV-DJ) containing N171-82Q mutant HTT (82Q) and N171-18Q wild type HTT (18Q; sham). The AAV-DJ-82Q model displayed motor dysfunctions in pole and rotarod tests beginning 4 weeks after viral infection in juvenile mice (8 weeks after birth). They showed behaviors reflecting neurodegeneration. They also showed increased apoptosis, robust glial activation and upregulated representative inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6), mediators (cyclooxygenase-2 and inducible nitric oxide synthase) and signaling pathways (nuclear factor kappa B and signal transducer and activator of transcription 3 (STAT3)) in the striatum at 10 weeks after viral infection (14 weeks after birth) via successful transfection of mutant HTT into neurons, microglia, and astrocytes in the striatum. However, little evidence of any of these events was found in mice infected with the AAV-DJ-18Q expressing construct. Intrastriatal injection of AAV-DJ-82Q might be useful as a novel in vivo model to investigate the biology of truncated N-terminal fragment (N171) in the striatum and to explore the efficacy of therapeutic strategies for HD. PMID:29946240

Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport

PubMed Central

Castle, Michael J.; Gershenson, Zachary T.; Giles, April R.; Holzbaur, Erika L.F.

2014-01-01

Abstract Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane

Adeno Associated Viral-mediated intraosseus labeling of bone marrow derived cells for CNS tracking

PubMed Central

Selenica, Maj-Linda B.; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B.; Nash, Kevin R.; Morgan, Dave; Gordon, Marcia N.; Lee, Daniel C.

2016-01-01

Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseus impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9–GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the

Transgene expression in target-defined neuron populations mediated by retrograde infection with adeno-associated viral vectors.

PubMed

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta; Wachowiak, Matt

2013-09-18

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

PubMed Central

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta

2013-01-01

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors—in particular, recombinant adeno-associated viral vectors (rAAVs)—have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested—in particular, though not exclusively, Cre-dependent vectors—showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal. PMID:24048849

Adeno-associated Virus as a Mammalian DNA Vector

PubMed Central

SALGANIK, MAX; HIRSCH, MATTHEW L.; SAMULSKI, RICHARD JUDE

2015-01-01

In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells. PMID:26350320

Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

PubMed

Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

2014-09-01

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice

PubMed Central

Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.

2005-01-01

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817

Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol

PubMed Central

Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

2016-01-01

Background Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. Objective The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. Methods We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. Results The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype

Novel Adeno-Associated Viral Vector Delivering the Utrophin Gene Regulator Jazz Counteracts Dystrophic Pathology in mdx Mice

PubMed Central

Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

2014-01-01

Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor “Jazz” that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. J. Cell. Physiol. 229: 1283–1291, 2014. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:24469912

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies.

PubMed

Sun, Junjiang; Hua, Baolai; Chen, Xiaojing; Samulski, Richard J; Li, Chengwen

2017-08-01

While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10 13 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

Analysis of Particle Content of Recombinant Adeno-Associated Virus Serotype 8 Vectors by Ion-Exchange Chromatography

PubMed Central

Lock, Martin; Alvira, Mauricio R.

2012-01-01

Abstract Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium. PMID:22428980

Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis.

PubMed

Mason, J B; Gurda, B L; Van Wettere, A; Engiles, J B; Wilson, J M; Richardson, D W

2017-01-01

Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Randomised in vivo experiment. We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the

Clades of Adeno-Associated Viruses Are Widely Disseminated in Human Tissues

PubMed Central

Gao, Guangping; Vandenberghe, Luk H.; Alvira, Mauricio R.; Lu, You; Calcedo, Roberto; Zhou, Xiangyang; Wilson, James M.

2004-01-01

The potential for using Adeno-associated virus (AAV) as a vector for human gene therapy has stimulated interest in the Dependovirus genus. Serologic data suggest that AAV infections are prevalent in humans, although analyses of viruses and viral sequences from clinical samples are extremely limited. Molecular techniques were used in this study to successfully detect endogenous AAV sequences in 18% of all human tissues screened, with the liver and bone marrow being the most predominant sites. Sequence characterization of rescued AAV DNAs indicated a diverse array of molecular forms which segregate into clades whose members share functional and serologic similarities. One of the most predominant human clades is a hybrid of two previously described AAV serotypes, while another clade was found in humans and several species of nonhuman primates, suggesting a cross-species transmission of this virus. These data provide important information regarding the biology of parvoviruses in humans and their use as gene therapy vectors. PMID:15163731

Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

PubMed

Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

2016-07-05

Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes

PubMed Central

Shu, Yilai; Tao, Yong; Wang, Zhengmin; Tang, Yong; Li, Huawei; Dai, Pu; Gao, Guangping; Chen, Zheng-Yi

2016-01-01

The mammalian inner ear consists of diverse cell types with important functions. Gene mutations in these diverse cell types have been found to underlie different forms of genetic hearing loss. Targeting these mutations for gene therapy development represents a future therapeutic strategy to treat hearing loss. Adeno-associated viral (AAV) vectors have become the vector of choice for gene delivery in animal models in vivo. To identify AAV vectors that target inner ear cell subtypes, we systemically screened 12 AAV vectors with different serotypes (AAV1, 2, 5, 6, 6.2, 7, 8, 9, rh.8, rh.10, rh.39, and rh.43) that carry a reporter gene GFP in neonatal and adult mice by microinjection in vivo. We found that most AAVs infect both neonatal and adult inner ear, with different specificities and expression levels. The inner ear cochlear sensory epithelial region, which includes auditory hair cells and supporting cells, is most frequently targeted for gene delivery. Expression of the transgene is sustained, and neonatal inner ear delivery does not adversely affect hearing. Adult inner ear injection of AAV has a similar infection pattern as the younger inner ear, with the exception that outer hair cell death caused by the injection procedure can lead to hearing loss. In the adult, more so than in the neonatal mice, cell types infected and efficiency of infection are correlated with the site of injection. Most infected cells survive in neonatal and adult inner ears. The study adds to the list of AAV vectors that transduce the mammalian inner ear efficiently, providing the tools that are important to study inner ear gene function and for the development of gene therapy to treat hearing loss. PMID:27342665

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

PubMed Central

Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V.; Nair, Sukesh C.; Srinivasan, Narayanaswamy; Srivastava, Alok

2013-01-01

Abstract Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (∼9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8

Recombination and Population Mosaic of a Multifunctional Viral Gene, Adeno-Associated Virus cap

PubMed Central

Takeuchi, Yasuhiro; Myers, Richard; Danos, Olivier

2008-01-01

Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV) cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI) revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u) region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred. PMID:18286191

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

PubMed

Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

2011-06-01

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

PubMed Central

Kilpatrick, Lauren A.; Li, Qian; Yang, John; Goddard, John C; Fekete, Donna M.; Lang, Hainan

2010-01-01

Murine models are ideal for studying cochlear gene transfer as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, due to its small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAV) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear and allows for near-complete preservation of low and middle frequency hearing. In the present study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6, and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus (CMV) promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells (IHCs) were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness. PMID:21209625

Adeno-associated virus-mediated gene transfer

PubMed Central

Srivastava, Arun

2008-01-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors in a murine serial bone marrow transplantation model in vivo, where stable integration of the proviral AAV genome does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. PMID:18500727

Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

2009-03-15

Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensivemore » than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.« less

[Ubiquitination of recombinant adeno-associated viral vector and its application].

PubMed

Wang, Qi-zhao; Lu, Ying-hui; Diao, Yong; Xu, Rui-an

2012-09-01

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

Adeno-associated virus-mediated gene transfer.

PubMed

Srivastava, Arun

2008-09-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

Engineering adeno-associated viruses for clinical gene therapy.

PubMed

Kotterman, Melissa A; Schaffer, David V

2014-07-01

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Engineering adeno-associated viruses for clinical gene therapy

PubMed Central

Kotterman, Melissa A.; Schaffer, David V.

2015-01-01

Clinical gene therapy has been increasingly successful, due both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among the latter, delivery vectors based on adeno-associated virus (AAV) have emerged as safe and effective – in one recent case leading to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers. PMID:24840552

Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells.

PubMed

Han, Zongchao; Zhong, Li; Maina, Njeri; Hu, Zhongbo; Li, Xiaomiao; Chouthai, Nitin S; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

2008-03-01

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

Adeno-Associated Viral-Mediated Catalase Expression Suppresses Optic Neuritis in Experimental Allergic Encephalomyelitis

NASA Astrophysics Data System (ADS)

Guy, John; Qi, Xiaoping; Hauswirth, William W.

1998-11-01

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Intracellular localization of adeno-associated viral proteins expressed in insect cells.

PubMed

Gallo-Ramírez, Lilí E; Ramírez, Octavio T; Palomares, Laura A

2011-01-01

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses ▿ †

PubMed Central

Grimm, Dirk; Lee, Joyce S.; Wang, Lora; Desai, Tushar; Akache, Bassel; Storm, Theresa A.; Kay, Mark A.

2008-01-01

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications. PMID:18400866

Molecular design for recombinant adeno-associated virus (rAAV) vector production.

PubMed

Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

2018-02-01

Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

PubMed

Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

1999-01-01

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

PubMed

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

2017-10-15

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

PubMed Central

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard

2017-01-01

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

PubMed

Kim, Eunmi; Oh, Ji-Seon; Ahn, Ik-Sung; Park, Kook In; Jang, Jae-Hyung

2011-11-01

Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.

2012-09-15

The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon,more » covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.« less

Single delivery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventricular tachycardia is able to cure the disease from birth to advanced age.

PubMed

Denegri, Marco; Bongianino, Rossana; Lodola, Francesco; Boncompagni, Simona; De Giusti, Verónica C; Avelino-Cruz, José E; Liu, Nian; Persampieri, Simone; Curcio, Antonio; Esposito, Francesca; Pietrangelo, Laura; Marty, Isabelle; Villani, Laura; Moyaho, Alejandro; Baiardi, Paola; Auricchio, Alberto; Protasi, Feliciano; Napolitano, Carlo; Priori, Silvia G

2014-06-24

Catecholaminergic polymorphic ventricular tachycardia is an inherited arrhythmogenic disorder characterized by sudden cardiac death in children. Drug therapy is still insufficient to provide full protection against cardiac arrest, and the use of implantable defibrillators in the pediatric population is limited by side effects. There is therefore a need to explore the curative potential of gene therapy for this disease. We investigated the efficacy and durability of viral gene transfer of the calsequestrin 2 (CASQ2) wild-type gene in a catecholaminergic polymorphic ventricular tachycardia knock-in mouse model carrying the CASQ2(R33Q/R33Q) (R33Q) mutation. We engineered an adeno-associated viral vector serotype 9 (AAV9) containing cDNA of CASQ2 wild-type (AAV9-CASQ2) plus the green fluorescent protein (GFP) gene to infect newborn R33Q mice studied by in vivo and in vitro protocols at 6, 9, and 12 months to investigate the ability of the infection to prevent the disease and adult R33Q mice studied after 2 months to assess whether the AAV9-CASQ2 delivery could revert the catecholaminergic polymorphic ventricular tachycardia phenotype. In both protocols, we observed the restoration of physiological expression and interaction of CASQ2, junctin, and triadin; the rescue of electrophysiological and ultrastructural abnormalities in calcium release units present in R33Q mice; and the lack of life-threatening arrhythmias. Our data demonstrate that viral gene transfer of wild-type CASQ2 into the heart of R33Q mice prevents and reverts severe manifestations of catecholaminergic polymorphic ventricular tachycardia and that this curative effect lasts for 1 year after a single injection of the vector, thus posing the rationale for the design of a clinical trial. © 2014 American Heart Association, Inc.

Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes

PubMed Central

Halbert, Christine L.; Rutledge, Elizabeth A.; Allen, James M.; Russell, David W.; Miller, A. Dusty

2000-01-01

Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors. PMID:10627564

Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

NASA Astrophysics Data System (ADS)

Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

2007-05-01

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

PubMed

Charbel Issa, Peter; De Silva, Samantha R; Lipinski, Daniel M; Singh, Mandeep S; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R; Hankins, Mark W; During, Matthew J; Maclaren, Robert E

2013-01-01

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/-) mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1) mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/-) mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

Structure of Adeno-Associated Virus Type 4

PubMed Central

Padron, Eric; Bowman, Valorie; Kaludov, Nikola; Govindasamy, Lakshmanan; Levy, Hazel; Nick, Phillip; McKenna, Robert; Muzyczka, Nicholas; Chiorini, John A.; Baker, Timothy S.; Agbandje-McKenna, Mavis

2005-01-01

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology. PMID:15795290

Convection Enhanced Delivery of Recombinant Adeno-associated Virus into the Mouse Brain.

PubMed

Nash, Kevin R; Gordon, Marcia N

2016-01-01

Recombinant adeno-associated virus (rAAV) has become an extremely useful tool for the study of gene over expression or knockdown in the central nervous system of experimental animals. One disadvantage of intracranial injections of rAAV vectors into the brain parenchyma has been restricted distribution to relatively small volumes of the brain. Convection enhanced delivery (CED) is a method for delivery of clinically relevant amounts of therapeutic agents to large areas of the brain in a direct intracranial injection procedure. CED uses bulk flow to increase the hydrostatic pressure and thus improve volume distribution. The CED method has shown robust gene transfer and increased distribution within the CNS and can be successfully used for different serotypes of rAAV for increased transduction of the mouse CNS. This chapter details the surgical injection of rAAV by CED into a mouse brain.

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

PubMed

Barajas, Daniel; Aponte-Ubillus, Juan Jose; Akeefe, Hassibullah; Cinek, Tomas; Peltier, Joseph; Gold, Daniel

2017-01-01

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

PubMed Central

Xia, Li; Yin, Shankai; Wang, Jian

2012-01-01

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis.

PubMed

van der Marel, Sander; Majowicz, Anna; Kwikkers, Karin; van Logtenstein, Richard; te Velde, Anje A; De Groot, Anne S; Meijer, Sybren L; van Deventer, Sander J; Petry, Harald; Hommes, Daniel W; Ferreira, Valerie

2012-08-28

-epithelial layer and the lamina propria. CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice, but were absent in the Tregitope 167 treated group. Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167. Furthermore, the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5 (cytomegalovirus promoter-Tregitope 167), as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice (P < 0.05 and P < 0.001, respectively). This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce long-term remission in inflammatory bowel disease.

Direct and Retrograde Transduction of Nigral Neurons with AAV6, 8, and 9 and Intraneuronal Persistence of Viral Particles

PubMed Central

Aebischer, Patrick

2013-01-01

Abstract Recombinant adeno-associated viral (AAV) vectors of serotypes 6, 8, and 9 were characterized as tools for gene delivery to dopaminergic neurons in the substantia nigra for future gene therapeutic applications in Parkinson's disease. While vectors of all three serotypes transduced nigral dopaminergic neurons with equal efficiency when directly injected to the substantia nigra, AAV6 was clearly superior to AAV8 and AAV9 for retrograde transduction of nigral neurons after striatal delivery. For sequential transduction of nigral dopaminergic neurons, the combination of AAV9 with AAV6 proved to be more powerful than AAV8 with AAV6 or repeated AAV6 administration. Surprisingly, single-stranded viral genomes persisted in nigral dopaminergic neurons within cell bodies and axon terminals in the striatum, and intact assembled AAV capsid was enriched in nuclei of nigral neurons, 4 weeks after virus injections to the substantia nigra. 6-Hydroxydopamine (6-OHDA)–induced degeneration of dopaminergic neurons in the substantia nigra reduced the number of viral genomes in the striatum, in line with viral genome persistence in axon terminals. However, 6-OHDA–induced axonal degeneration did not induce any transsynaptic spread of AAV infection in the striatum. Therefore, the potential presence of viral particles in axons may not represent an important safety issue for AAV gene therapy applications in neurodegenerative diseases. PMID:23600720

Adeno-associated virus vector-mediated transduction in the cat brain.

PubMed

Vite, Charles H; Passini, Marco A; Haskins, Mark E; Wolfe, John H

2003-10-01

Adeno-associated virus (AAV) vectors are capable of delivering a therapeutic gene to the mouse brain that can result in long-term and widespread protein production. However, the human infant brain is more than 1000 times larger than the mouse brain, which will make the treatment of global neurometabolic disorders in children more difficult. In this study, we evaluated the ability of three AAV serotypes (1,2, and 5) to transduce cells in the cat brain as a model of a large mammalian brain. The human lysosomal enzyme beta-glucuronidase (GUSB) was used as a reporter gene, because it can be distinguished from feline GUSB by heat stability. The vectors were injected into the cerebral cortex, caudate nucleus, thalamus, corona radiata, internal capsule, and centrum semiovale of 8-week-old cats. The brains were evaluated for gene expression using in situ hybridization and enzyme histochemistry 10 weeks after surgery. The AAV2 vector was capable of transducing cells in the gray matter, while the AAV1 vector resulted in greater transduction of the gray matter than AAV2 as well as transduction of the white matter. AAV5 did not result in detectable transduction in the cat brain.

Recombinant avian adeno-associated virus: transgene expression in vivo and enhancement of expression in vitro.

PubMed

Estevez, Carlos; Villegas, Pedro

2006-06-01

Recombinant avian adeno-associated viruses coding for the LacZ gene were used to inoculate embryonating chicken eggs, to assess the usefulness of the system for the expression of a transgene in vivo. The results obtained indicate significantly higher levels of expression of the reporter gene at various time intervals in the embryos inoculated with the recombinant virus in comparison with the mock-inoculated controls. At the embryo level, significant differences were evident at 120 hr postinoculation; hatched chicks showed transgene expression up to 14 days of age. In a second experiment, different cell-line cultures were transfected with plasmids encoding for a reporter gene flanked by the avian adeno-associated virus inverted terminal repeats (ITR), either alone or in the presence of the major nonstructural proteins of the virus (Rep 78/68) to assess the ability of these proteins and DNA elements to enhance gene expression. Results indicate that the inclusion of the viral ITR alone or during coexpression of the Rep proteins significantly enhances the expression of the transgene in all cell lines tested, as evidenced by the detection of the beta-galacrosidase protein through chemiluminescence reactions and staining of transfected monolayers.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors.

PubMed

Vande Velde, G; Rangarajan, J R; Toelen, J; Dresselaers, T; Ibrahimi, A; Krylychkina, O; Vreys, R; Van der Linden, A; Maes, F; Debyser, Z; Himmelreich, U; Baekelandt, V

2011-06-01

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Detectable reporter gene expression following transduction of adenovirus and adeno-associated virus serotype 2 vectors within full-thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo.

PubMed

Santangelo, Kelly S; Baker, Sarah A; Nuovo, Gerard; Dyce, Jonathan; Bartlett, Jeffrey S; Bertone, Alicia L

2010-02-01

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. (c) 2009 Orthopaedic Research Society.

Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

PubMed Central

Hammond, Sean L.; Leek, Ashley N.; Richman, Evan H.

2017-01-01

The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression in vivo and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined in vivo at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson’s Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain

Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences.

PubMed

Nony, P; Tessier, J; Chadeuf, G; Ward, P; Giraud, A; Dugast, M; Linden, R M; Moullier, P; Salvetti, A

2001-10-01

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.

PubMed

Marsic, Damien; Méndez-Gómez, Héctor R; Zolotukhin, Sergei

2015-01-01

Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

PubMed

Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

2007-05-01

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Stent-based delivery of adeno-associated viral vectors with sustained vascular transduction and iNOS-mediated inhibition of in-stent restenosis

PubMed Central

Fishbein, Ilia; Guerrero, David T.; Alferiev, Ivan S.; Foster, Jonathan B.; Minutolo, Nicholas G.; Chorny, Michael; Mas Monteys, Alejandro; Driesbaugh, Kathryn H.; Nagaswami, Chandrasekaran; Levy, Robert J.

2017-01-01

In-stent restenosis remains an important clinical problem in the era of drug eluting stents. Development of clinical gene therapy protocols for the prevention and treatment of in-stent restenosis is hampered by the lack of adequate local delivery systems. Herein we describe a novel stent-based gene delivery platform capable of providing local arterial gene transfer with adeno-associated viral (AAV) vectors. This system exploits the natural affinity of protein G (PrG) to bind to the Fc region of mammalian IgG, making PrG a universal adaptor for surface immobilization of vector-capturing antibodies (Ab). Our results: 1) demonstrate the feasibility of reversible immobilization of AAV2 vectors using vector tethering by AAV2-specific Ab appended to the stent surface through covalently attached PrG, 2) show sustained release kinetics of PrG/Ab-immobilized AAV2 vector particles into simulated physiological medium in vitro and site-specific transduction of cultured cells, 3) provide evidence of long-term (12 weeks) arterial expression of luciferase with PrG/Ab-tethered AAV2Luc, and 4) show anti-proliferative activity and anti-restenotic efficacy of stent-immobilized AAV2iNOS in the rat carotid artery model of stent angioplasty. PMID:28832561

Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

PubMed Central

Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

2011-01-01

Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes

PubMed Central

Chan, Kuan Rong; Tan, Hwee Cheng; Bestagno, Marco; Ooi, Eng Eong; Burrone, Oscar R.

2015-01-01

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well. PMID:26218926

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

PubMed Central

Zhang, Wenli; Solanki, Manish; Müther, Nadine; Ebel, Melanie; Wang, Jichang; Sun, Chuanbo; Izsvak, Zsuzsanna; Ehrhardt, Anja

2013-01-01

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells. PMID:24116154

Myocardial Adeno-Associated Virus Serotype 6–βARKct Gene Therapy Improves Cardiac Function and Normalizes the Neurohormonal Axis in Chronic Heart Failure

PubMed Central

Rengo, Giuseppe; Lymperopoulos, Anastasios; Zincarelli, Carmela; Donniacuo, Maria; Soltys, Stephen; Rabinowitz, Joseph E.; Koch, Walter J.

2009-01-01

Background The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function. The peptide βARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve βAR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term βARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results In HF rats, we delivered βARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the β-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. βARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac βAR signaling. Addition of metoprolol neither enhanced nor decreased βARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions Long-term cardiac AAV6-βARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, βARKct alone improves outcomes more than a β-blocker alone, whereas both treatments are compatible. These findings show that βARKct gene therapy can be of long-term therapeutic value in HF. PMID:19103992

Mapping and Engineering Functional Domains of the Assembly Activating Protein of Adeno-Associated Viruses.

PubMed

Tse, Longping V; Moller-Tank, Sven; Meganck, Rita M; Asokan, Aravind

2018-04-25

Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis followed by deletion and substitutional mutagenesis of specific domains, namely, the hydrophobic N-terminal domain (HR), conserved core (CC), proline-rich region (PRR), threonine/serine rich region (T/S) and basic region (BR). First, we establish that the centrally located PRR and T/S regions are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display varying ability to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N-terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful towards understanding and controlling AAV capsid assembly. Importance: Adeno-associated viruses (AAV) encode a unique assembly activating protein (AAP) within their genome that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a

Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction

PubMed Central

Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude

2016-01-01

Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

PubMed

Asavarut, Paladd; O'Neill, Kevin; Syed, Nelofer; Hajitou, Amin

2014-01-01

The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil.

PubMed

Marinho, Paula Eillanny Silva; Bretas de Oliveira, Danilo; Candiani, Talitah Michel Sanchez; Crispim, Ana Paula Correia; Alvarenga, Pedro Paulo Martins; Castro, Fabrizia Cristina Dos Santos; Abrahão, Jonatas Santos; Rios, Maria; Coimbra, Roney Santos; Kroon, Erna Geessien

2017-01-01

To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014-2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2.

[Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities].

PubMed

Grinenko, N F; Savitskaia, N V; Pashvykina, G V; Al'tshteĭn, A D

2003-06-01

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.

PubMed

Piedra, Jose; Ontiveros, Maria; Miravet, Susana; Penalva, Cristina; Monfar, Mercè; Chillon, Miguel

2015-02-01

Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles.

PubMed

Smith, J Kennon; Agbandje-McKenna, Mavis

2018-05-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding ("footprints") can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein-DNA and protein-protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle-specifically, those involved with binding neutralizing antibodies (NAbs) [2-4]. This method offers a unique opportunity for visualizing antibody binding "hotspots" on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create "stealth" AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms.

Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles

PubMed Central

Agbandje-McKenna, Mavis

2018-01-01

The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding (“footprints”) can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein–DNA and protein–protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle—specifically, those involved with binding neutralizing antibodies (NAbs) [2–4]. This method offers a unique opportunity for visualizing antibody binding “hotspots” on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create “stealth” AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms. PMID:29723270

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

PubMed Central

Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

2009-01-01

Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

Adeno-associated virus type 2 binding study on model heparan sulfate surface

NASA Astrophysics Data System (ADS)

Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

2003-11-01

Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

PubMed Central

Adamson-Small, Laura; Potter, Mark; Byrne, Barry J.; Clément, Nathalie

2017-01-01

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 1015 VG in crude cell harvests and an average of 3.5 × 1014 total VG of purified rAAV9 material, resulting in greater than 1 × 105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production. PMID:28117600

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

PubMed

Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J; Castellanos Rivera, Ruth M; Wells, Lindsey F; Havlik, L Patrick; Smith, J Kennon; Agbandje-McKenna, Mavis; Asokan, Aravind

2017-06-13

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

Adeno-associated virus rep protein synthesis during productive infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Redemann, B.E.; Mendelson, E.; Carter, B.J.

1989-02-01

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing tomore » a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.« less

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

PubMed

Kyostio-Moore, Sirkka; Berthelette, Patricia; Cornell, Cathleen Sookdeo; Nambiar, Bindu; Figueiredo, Monica Dias

2018-05-01

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.

PubMed

Ohi, S

2000-07-01

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research.

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

PubMed

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Association between nasopharyngeal load of Streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children.

PubMed

Vu, Huong Thi Thu; Yoshida, Lay Myint; Suzuki, Motoi; Nguyen, Hien Anh Thi; Nguyen, Cat Dinh Lien; Nguyen, Ai Thi Thuy; Oishi, Kengo; Yamamoto, Takeshi; Watanabe, Kiwao; Vu, Thiem Dinh

2011-01-01

The interplay between nasopharyngeal bacterial carriage, viral coinfection, and lower respiratory tract infections (LRTIs) is poorly understood. We explored this association in Vietnamese children aged less than 5 years. A hospital-based case-control study of pediatric LRTIs was conducted in Nha Trang, Vietnam. A total of 550 hospitalized children (274 radiologically confirmed pneumonia [RCP] and 276 other LRTIs) were enrolled and 350 healthy controls were randomly selected from the community. Polymerase chain reaction-based methods were used to measure bacterial loads of Streptococcus pneumoniae (SP), Haemophilus influenzae, and Moraxella catarrhalis and to detect 13 respiratory viruses and bacterial serotypes in nasopharyngeal samples of study participants. The median nasopharyngeal bacterial load of SP was substantially higher in children with RCP compared with healthy controls or children with other LRTIs (P < 0.001). SP load was 15-fold higher in pneumonia children with viral coinfection compared with those children without viral coinfection (1.4 x 10⁷/mL vs. 9.1 x 10⁵/mL; P 0.0001). SP load was over 200-fold higher in serotypeable SP compared with nontypeable SP (2.5 x 10⁶/mL vs. 1 x 10⁴/mL; P < 0.0001). These associations were independent of potential confounders in multiple regression models. No clear association was found between nasopharyngeal load of Haemophilus influenzae or Moraxella catarrhalis and viral coinfection in either RCP or other LRTIs groups. An increased load of SP in the nasopharynx was associated with RCP, viral coinfection, and presence of pneumococcal capsule.

Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy.

PubMed

Zygmunt, Deborah A; Crowe, Kelly E; Flanigan, Kevin M; Martin, Paul T

2017-09-01

Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (<18 years old) had measurable rAAV titers to all serotypes tested, and this percentage increased to almost 50% in adult normal subjects (>18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.

Adeno-associated viral gene therapy for mucopolysaccharidoses exhibiting neurodegeneration.

PubMed

Lau, Adeline A; Hemsley, Kim M

2017-10-01

The mucopolysaccharidoses (MPS) are a subgroup of lysosomal storage disorders that are caused by mutations in the genes involved in glycosaminoglycan breakdown. Multiple organs and tissues are affected, including the central nervous system. At present, hematopoietic stem cell transplantation and enzyme replacement therapies are approved for some of the (non-neurological) MPS. Treatments that effectively ameliorate the neurological aspects of the disease are being assessed in clinical trials. This review will focus on the recent outcomes and planned viral vector-mediated gene therapy clinical trials, and the pre-clinical data that supported these studies, for MPS-I (Hurler/Scheie syndrome), MPS-II (Hunter syndrome), and MPS-IIIA and -IIIB (Sanfilippo syndrome).

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

MRI-Guided Delivery of Viral Vectors.

PubMed

Salegio, Ernesto A; Bringas, John; Bankiewicz, Krystof S

2016-01-01

Gene therapy has emerged as a potential avenue of treatment for many neurological disorders. Technological advances in imaging techniques allow for the monitoring of real-time infusions into the brain of rodents, nonhuman primates, and humans. Here, we discuss the use of magnetic resonance imaging (MRI) as a tool in the delivery of adeno-associated viral (AAV) particles into brain of nonhuman primates.

Conformational changes in intact dengue virus reveal serotype-specific expansion

PubMed Central

Lim, Xin-Xiang; Chandramohan, Arun; Lim, Xin Ying Elisa; Bag, Nirmalya; Sharma, Kamal Kant; Wirawan, Melissa; Wohland, Thorsten; Lok, Shee-Mei; Anand, Ganesh S.

2017-01-01

Dengue virus serotype 2 (DENV2) alone undergoes structural expansion at 37 °C (associated with host entry), despite high sequence and structural homology among the four known serotypes. The basis for this differential expansion across strains and serotypes is unknown and necessitates mapping of the dynamics of dengue whole viral particles to describe their coordinated motions and conformational changes when exposed to host-like environments. Here we capture the dynamics of intact viral particles of two serotypes, DENV1 and DENV2, by amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and time resolved Förster Resonance Energy Transfer. Our results show temperature-dependent dynamics hotspots on DENV2 and DENV1 particles with DENV1 showing expansion at 40 °C but not at 37 °C. HDXMS measurement of virion dynamics in solution offers a powerful approach to identify potential epitopes, map virus-antibody complex structure and dynamics, and test effects of multiple host-specific perturbations on viruses and virus-antibody complexes. PMID:28186093

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

PubMed

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

High-Efficiency Promoter-Dependent Transduction by Adeno-Associated Virus Type 6 Vectors in Mouse Lung

PubMed Central

HALBERT, CHRISTINE L.; LAM, SIU-LING; MILLER, A. DUSTY

2014-01-01

The transduction efficiency of adeno-associated virus (AAV) vectors in various somatic tissues has been shown to depend heavily on the AAV type from which the vector capsid proteins are derived. Among the AAV types studied, AAV6 efficiently transduces cells of the airway epithelium, making it a good candidate for the treatment of lung diseases such as cystic fibrosis. Here we have evaluated the effects of various promoter sequences on transduction rates and gene expression levels in the lung. Of the strong viral promoters examined, the Rous sarcoma virus (RSV) promoter performed significantly better than a human cytomegalovirus (CMV) promoter in the airway epithelium. However, a hybrid promoter consisting of a CMV enhancer, β-actin promoter and splice donor, and a β-globin splice acceptor (CAG promoter) exhibited even higher expression than either of the strong viral promoters alone, showing a 38-fold increase in protein expression over the RSV promoter. In addition, we show that vectors containing either the RSV or CAG promoter expressed well in the nasal and tracheal epithelium. Transduction rates in the 90% range were achieved in many airways with the CAG promoter, showing that with the proper AAV capsid proteins and promoter sequences, highly efficient transduction can be achieved. PMID:17430088

Toxoplasma serotype is associated with development of ocular toxoplasmosis.

PubMed

Shobab, Leila; Pleyer, Uwe; Johnsen, Joerdis; Metzner, Sylvia; James, Erick R; Torun, N; Fay, Michael P; Liesenfeld, Oliver; Grigg, Michael E

2013-11-01

Worldwide, ocular toxoplasmosis (OT) is the principal cause of posterior uveitis, a severe, life-altering disease. A Toxoplasma gondii enzyme-linked immunoassay that detects strain-specific antibodies present in serum was used to correlate serotype with disease. Toxoplasma serotypes in consecutive serum samples from German uveitis patients with OT were compared with non-OT seropositive patients with noninfectious autoimmune posterior uveitis. OT patients were tested for association of parasite serotype with age, gender, location, clinical onset, size, visual acuity, or number of lesions (mean follow-up, 3.8 years) to determine association with recurrences. A novel, nonreactive (NR) serotype was detected more frequently in serum samples of OT patients (50/114, 44%) than in non-OT patients (4/56, 7%) (odds ratio, 10.0; 95% confidence interval 3.4-40.8; P < .0001). Non-OT patients were predominantly infected with Type II strains (39/56; 70%), consistent with expected frequencies in Central Europe. Among OT patients, those with NR serotypes experienced more frequent recurrences (P = .037). Polymerase chain reaction detected parasite DNA in 8/60 OT aqueous humor specimens but failed to identify Type II strain alleles. Toxoplasma NR and Type II serotypes predominate in German OT patients. The NR serotype is associated with OT recurrences, underscoring the value of screening for management of disease.

Regulation of adeno-associated virus DNA replication by the cellular TAF-I/set complex.

PubMed

Pegoraro, Gianluca; Marcello, Alessandro; Myers, Michael P; Giacca, Mauro

2006-07-01

The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.

Methods of treating Parkinson's disease using viral vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bankiewicz, Krystof; Cunningham, Janet

Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson's Disease.

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

PubMed

Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

2016-02-01

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yakobson, B.; Hrynko, T.A.; Peak, M.J.

1989-03-01

Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting amore » requirement for de novo protein synthesis.« less

Toxoplasma Serotype Is Associated With Development of Ocular Toxoplasmosis

PubMed Central

Shobab, Leila; Pleyer, Uwe; Johnsen, Joerdis; Metzner, Sylvia; James, Erick R.; Torun, N.; Fay, Michael P.; Liesenfeld, Oliver; Grigg, Michael E.

2013-01-01

Background. Worldwide, ocular toxoplasmosis (OT) is the principal cause of posterior uveitis, a severe, life-altering disease. A Toxoplasma gondii enzyme-linked immunoassay that detects strain-specific antibodies present in serum was used to correlate serotype with disease. Methods. Toxoplasma serotypes in consecutive serum samples from German uveitis patients with OT were compared with non-OT seropositive patients with noninfectious autoimmune posterior uveitis. OT patients were tested for association of parasite serotype with age, gender, location, clinical onset, size, visual acuity, or number of lesions (mean follow-up, 3.8 years) to determine association with recurrences. Results. A novel, nonreactive (NR) serotype was detected more frequently in serum samples of OT patients (50/114, 44%) than in non-OT patients (4/56, 7%) (odds ratio, 10.0; 95% confidence interval 3.4–40.8; P < .0001). Non-OT patients were predominantly infected with Type II strains (39/56; 70%), consistent with expected frequencies in Central Europe. Among OT patients, those with NR serotypes experienced more frequent recurrences (P = .037). Polymerase chain reaction detected parasite DNA in 8/60 OT aqueous humor specimens but failed to identify Type II strain alleles. Conclusions. Toxoplasma NR and Type II serotypes predominate in German OT patients. The NR serotype is associated with OT recurrences, underscoring the value of screening for management of disease. PMID:23878321

Assaying the Stability and Inactivation of AAV Serotype 1 Vectors

PubMed Central

Howard, Douglas B.; Harvey, Brandon K.

2017-01-01

Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze–thaw cycles, the resulting transduction efficiency became variable at 60–120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments. PMID:28192678

Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

NASA Astrophysics Data System (ADS)

Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

1997-12-01

About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

Transduction of Nonhuman Primate Brain with Adeno-Associated Virus Serotype 1: Vector Trafficking and Immune Response

PubMed Central

Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L.; Bankiewicz, Krystof S.

2009-01-01

Abstract We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohisto-chemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm3 coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4+ lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2. PMID:19292604

Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

PubMed

Bell, Peter; Vandenberghe, Luk H; Wilson, James M

2014-06-01

Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

Production and validation of recombinant adeno-associated virus for channelrhodopsin expression in neurons.

PubMed

Lin, John Y

2013-01-01

Recent discovery of the light-activated ion channel, channelrhodopsin (ChR), has provided researchers a powerful and convenient tool to manipulate the membrane potential of specific cells with light. With genetic targeting of these channels and illumination of light to a specific location, the experimenter can selectively activate the voltage-gated ion channels (VGICs) of ChR-expressing cells, initiating electrical signaling in temporally and spatially precise manners. In neuroscience research, this can be used to study electrical signal processing within one neuron at the cellular level, or the synaptic connectivity between neurons at the circuitry level. To conduct experiments with ChRs, these exogenous channels need to be introduced into the cells of interest, commonly through a viral approach. This chapter provides an overview of the design, production, and validation of recombinant adeno-associated virus (rAAV) for ChR expression that can be used in vitro or in vivo to infect neurons. The virus produced can be used to conduct "optogenetic" experiments in behaving animals, in vitro preparations and cultured cells, and can be used to study signal transduction and processing at a cellular or circuitry level.

Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

PubMed

Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

2013-01-01

Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

PubMed Central

Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

2014-01-01

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

Are risk factors associated with invasive pneumococcal disease according to different serotypes?

PubMed

Ciruela, Pilar; Soldevila, Núria; Selva, Laura; Hernández, Sergi; Garcia-Garcia, Juan Jose; Moraga, Fernando; de Sevilla, Mariona F; Codina, Gemma; Planes, Ana Maria; Esteva, Cristina; Coll, Francis; Cardeñosa, Neus; Jordan, Iolanda; Batalla, Joan; Salleras, Luis; Muñoz-Almagro, Carmen; Domínguez, Angela

2013-03-01

The aim of this study was to investigate risk factors for the most common serotypes of invasive pneumococcal disease (IPD). A total of 293 IPD cases were analyzed in children aged 3-59 mo in a community with intermediate vaccination coverage with the 7-valent pneumococcal vaccine (PCV7). IPD cases were reviewed during 2007-2009 in two pediatric hospitals in Catalonia (Spain). A multivariate analysis using unconditional logistic regression was performed to estimate the adjusted odds ratio. PCV7 coverage was 45.4%. Pneumonia with empyema (64.5%) was the most frequent clinical manifestation. The most common serotypes were: serotype 1 (21.2%), 19A (16.0%), 3 (12.6%) and 7F/A (6.8%). 70.0% of serotypes found were included in the 13-valent conjugate vaccine (PCV13), 39.2% in the 10-valent conjugate vaccine and 8.1% in the PCV7. PCV7 was protective in IPD cases due to PCV7-serotypes (aOR: 0.15, 95% CI:0.04-0.55). Serotype 1 was positively associated with attending day care or school (aOR: 3.55, 95% CI: 1.21-10.38) and age 24-59 mo (aOR: 7.70, 95% CI:2.70-21.98). Serotype 19A was positively associated with respiratory infection in the previous month (aOR: 2.26, 95% CI: 1.03-4.94), non-penicillin susceptible IPD (aOR: 1.89, 95% CI:1.13-3.16) and negatively associated with age 24-59 mo (aOR: 0.19, 95% CI:0.09-0.41). Serotype 3 was positively associated with vaccination (aOR: 4.87, 95% CI:2.05-11.59). No factors were associated with serotype 7F/A. Vaccination with pneumococcal vaccines including more serotypes may reduce the risk of disease in our setting.

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

PubMed

White, K; Büning, H; Kritz, A; Janicki, H; McVey, J; Perabo, L; Murphy, G; Odenthal, M; Work, L M; Hallek, M; Nicklin, S A; Baker, A H

2008-03-01

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Are risk factors associated with invasive pneumococcal disease according to different serotypes?

PubMed Central

Ciruela, Pilar; Soldevila, Núria; Selva, Laura; Hernández, Sergi; Garcia-Garcia, Juan Jose; Moraga, Fernando; de Sevilla, Mariona F.; Codina, Gemma; Planes, Ana Maria; Esteva, Cristina; Coll, Francis; Cardeñosa, Neus; Jordan, Iolanda; Batalla, Joan; Salleras, Luis; Muñoz-Almagro, Carmen; Domínguez, Angela

2013-01-01

The aim of this study was to investigate risk factors for the most common serotypes of invasive pneumococcal disease (IPD). A total of 293 IPD cases were analyzed in children aged 3–59 mo in a community with intermediate vaccination coverage with the 7-valent pneumococcal vaccine (PCV7). IPD cases were reviewed during 2007–2009 in two pediatric hospitals in Catalonia (Spain). A multivariate analysis using unconditional logistic regression was performed to estimate the adjusted odds ratio. PCV7 coverage was 45.4%. Pneumonia with empyema (64.5%) was the most frequent clinical manifestation. The most common serotypes were: serotype 1 (21.2%), 19A (16.0%), 3 (12.6%) and 7F/A (6.8%). 70.0% of serotypes found were included in the 13-valent conjugate vaccine (PCV13), 39.2% in the 10-valent conjugate vaccine and 8.1% in the PCV7. PCV7 was protective in IPD cases due to PCV7-serotypes (aOR: 0.15, 95% CI:0.04–0.55). Serotype 1 was positively associated with attending day care or school (aOR: 3.55, 95% CI: 1.21–10.38) and age 24–59 mo (aOR: 7.70, 95% CI:2.70–21.98). Serotype 19A was positively associated with respiratory infection in the previous month (aOR: 2.26, 95% CI: 1.03–4.94), non-penicillin susceptible IPD (aOR: 1.89, 95% CI:1.13–3.16) and negatively associated with age 24–59 mo (aOR: 0.19, 95% CI:0.09–0.41). Serotype 3 was positively associated with vaccination (aOR: 4.87, 95% CI:2.05–11.59). No factors were associated with serotype 7F/A. Vaccination with pneumococcal vaccines including more serotypes may reduce the risk of disease in our setting. PMID:23295982

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

PubMed

Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

2009-11-01

Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

My Life with Adeno-Associated Virus: A Long Time Spent Studying a Short Genome

PubMed Central

2013-01-01

My 45 years of studying the molecular biology of adeno-associated virus are recounted. Additional activities as a mentor, department chair, and medical school administrator are described, as are my activities in the public sphere, which involved national issues related to science policy and medical education. PMID:23781880

Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.

2006-07-15

Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months inmore » plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.« less

Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cassell, Geoffrey D.; Weitzman, Matthew D.

2004-10-01

Adeno-associated virus (AAV) replicates in the nucleus of infected cells, and therefore multiple nuclear import events are required for productive infection. We analyzed nuclear import of the viral Rep proteins and characterized a nuclear localization signal (NLS) in the C-terminus. We demonstrate that basic residues in this region constitute an NLS that is transferable and mediates interaction with the nuclear import receptor importin {alpha} in vitro. Mutant Rep proteins are predominantly cytoplasmic and are severely compromised for interactions with importin {alpha}, but retain their enzymatic functions in vitro. Interestingly, mutations of the NLS had significantly less effect on importin {alpha}more » interaction and replication in the context of Rep78 than when incorporated into the Rep68 protein. Together, our results demonstrate that a bipartite NLS exists in the shared part of Rep68 and Rep78, and suggest that an alternate entry mechanism may also contribute to nuclear localization of the Rep78 protein.« less

Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

PubMed Central

Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Kekilli, Demet; Samso, Monserrat

2013-01-01

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle. PMID:23152528

Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage*

PubMed Central

Stoneham, Charlotte A.; Hollinshead, Michael; Hajitou, Amin

2012-01-01

Adeno-associated virus/phage (AAVP) is a gene delivery vector constructed as a hybrid between adeno-associated virus and filamentous phage. Tumor targeting following systemic administration has previously been demonstrated in several in vivo cancer models, with tumor specificity achieved through display of an αv integrin-targeting ligand on the capsid. However, high titers of AAVP are required for transduction of large numbers of mammalian cells. This study is the first to investigate the mechanisms involved in entry and intracellular trafficking of AAVP. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, we have demonstrated that targeted AAVP endocytosis is both dynamin and clathrin-dependent. Following entry, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway. Finally, we have demonstrated that disruption of this pathway leads to improved transgene expression by AAVP, thus demonstrating that escape from the late endosomes/lysosomes is a critical step for improving gene delivery by AAVP. These findings have important implications for the rational design of improved AAVP and RGD-targeted vectors. PMID:22915587

The prevalence of dengue virus serotypes in asymptomatic blood donors reveals the emergence of serotype 4 in Saudi Arabia.

PubMed

Ashshi, Ahmed Mohamed

2017-06-09

Transmission of dengue virus (DENV) through blood transfusion has been documented and hence screening for DENV during blood donation has been recently recommended by the American Association of Blood Banks and Centres of Disease Control and Prevention. DENV is endemic in the Western province of the Kingdom of Saudi Arabia (KSA) and serotypes 1, 2 and 3, but not 4, have been detected. However, little is known regarding the rates of DENV during blood donation in the kingdom. The aim of this study was therefore to measure the prevalence of dengue virus and its serotypes in eligible Saudi blood donors in the endemic Western region of KSA. This was a cross-sectional study and serum samples were collected from 910 eligible Saudi male blood donors. DENV IgM and IgG antibodies were measured serologically by ELISA while viral serotypes were detected by a single step IVD CE certified multiplex RT-PCR kit. The overall prevalence was 39 and 5.5% for IgG+ and IgM+, respectively. There were 12 (1.3%) with exclusively IgM+, 317 (34.8%) exclusively IgG+ and 38 (4.2%) with dual IgM+/IgG+ donors. The overall prevalence was 3.2% (n = 29) and 2.3% (n = 21) for primary and secondary infections. PCR was positive in 5.5% (n = 50) and, DENV-2 (n = 24; 48%) was the most frequent serotype and was significantly higher than DENV-1 (20%; P = 0.02) and DENV-3 (2%; P = 0.1 × 10 -5 ) but not DENV-4 (30%; P = 0.2). There was no significant difference between both DENV-4 and DENV-1 (P = 0.4). The combination of the PCR and serology findings showed that 22 (2.4%) and 28 (3.1%) donors had primary and secondary viremic infections, respectively. The detected rates of DENV by PCR suggest a potential high risk of viral transmission by blood transfusion. To the best of our knowledge, this study is the first to report the detection of DENV-4 serotype in Saudi Arabia. More studies are required to measure the precise prevalence of DENV serotypes and their potential

Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

PubMed

Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

2016-02-01

Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Correction of the enzymatic and functional deficits in a model of Pompe disease using adeno-associated virus vectors.

PubMed

Fraites, Thomas J; Schleissing, Mary R; Shanely, R Andrew; Walter, Glenn A; Cloutier, Denise A; Zolotukhin, Irene; Pauly, Daniel F; Raben, Nina; Plotz, Paul H; Powers, Scott K; Kessler, Paul D; Byrne, Barry J

2002-05-01

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

PubMed Central

Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

2015-01-01

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

Adeno-associated virus transfer of a gene encoding SNAP-25 resistant to botulinum toxin A attenuates neuromuscular paralysis associated with botulism.

PubMed

Raghunath, Arvind; Perez-Branguli, Francesc; Smith, Leonard; Dolly, J Oliver

2008-04-02

Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

Outbreak-associated Salmonella enterica Serotypes and Food Commodities, United States, 1998–2008

PubMed Central

Griffin, Patricia M.; Cole, Dana; Walsh, Kelly A.; Chai, Shua J.

2013-01-01

Salmonella enterica infections are transmitted not only by animal-derived foods but also by vegetables, fruits, and other plant products. To clarify links between Salmonella serotypes and specific foods, we examined the diversity and predominance of food commodities implicated in outbreaks of salmonellosis during 1998–2008. More than 80% of outbreaks caused by serotypes Enteritidis, Heidelberg, and Hadar were attributed to eggs or poultry, whereas >50% of outbreaks caused by serotypes Javiana, Litchfield, Mbandaka, Muenchen, Poona, and Senftenberg were attributed to plant commodities. Serotypes Typhimurium and Newport were associated with a wide variety of food commodities. Knowledge about these associations can help guide outbreak investigations and control measures. PMID:23876503

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

Restoration of central nervous system alpha-N-acetylglucosaminidase activity and therapeutic benefits in mucopolysaccharidosis IIIB mice by a single intracisternal recombinant adeno-associated viral type 2 vector delivery.

PubMed

Fu, Haiyan; DiRosario, Julianne; Kang, Lu; Muenzer, Joseph; McCarty, Douglas M

2010-07-01

Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction. In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease. We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues. A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael

2005-05-10

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, couldmore » be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.« less

Characterization of Interactions between Heparin/Glycosaminoglycan and Adeno-associated Virus

PubMed Central

Zhang, Fuming; Aguilera, Javier; Beaudet, Julie M.; Xie, Qing; Lerch, Thomas F.; Davulcu, Omar; Colón, Wilfredo; Chapman, Michael S.; Linhardt, Robert J.

2013-01-01

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this report, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans. Surface plasmon resonance results revealed that heparin binds to AAV with extremely high affinity. Solution competition studies shows that AAV binding to heparin is chain length dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV- heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 complexed with heparin. PMID:23952613

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids

PubMed Central

Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

2016-01-01

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration—at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then—through a variety of mechanisms—result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections.

PubMed

Robert, Marc-André; Nassoury, Nasha; Chahal, Parminder S; Venne, Marie-Hélène; Racine, Trina; Qiu, Xiangguo; Kobinger, Gary; Kamen, Amine; Gilbert, Rénald; Gaillet, Bruno

2018-04-01

Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 10 10 and 13.0 × 10 10 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 10 10 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.

Strategies for targeting primate neural circuits with viral vectors

PubMed Central

El-Shamayleh, Yasmine; Ni, Amy M.

2016-01-01

Understanding how the brain works requires understanding how different types of neurons contribute to circuit function and organism behavior. Progress on this front has been accelerated by optogenetics and chemogenetics, which provide an unprecedented level of control over distinct neuronal types in small animals. In primates, however, targeting specific types of neurons with these tools remains challenging. In this review, we discuss existing and emerging strategies for directing genetic manipulations to targeted neurons in the adult primate central nervous system. We review the literature on viral vectors for gene delivery to neurons, focusing on adeno-associated viral vectors and lentiviral vectors, their tropism for different cell types, and prospects for new variants with improved efficacy and selectivity. We discuss two projection targeting approaches for probing neural circuits: anterograde projection targeting and retrograde transport of viral vectors. We conclude with an analysis of cell type-specific promoters and other nucleotide sequences that can be used in viral vectors to target neuronal types at the transcriptional level. PMID:27052579

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

PubMed Central

Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

2009-01-01

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

New insights on infectious bronchitis virus pathogenesis: characterization of Italy 02 serotype in chicks and adult hens.

PubMed

Dolz, Roser; Vergara-Alert, Júlia; Pérez, Mónica; Pujols, Joan; Majó, Natàlia

2012-05-04

Infectious bronchitis (IB) is a worldwide disease affecting chickens of all ages and causing important economic losses in poultry industry. Despite being one of the predominant IB virus (IBV) serotype in several European countries, slightly is known about pathogenesis and pathogenicity of Italy 02 serotype. In this study chicks and old hens were infected by oculo-nasal route with Italy 02 serotype. Clinical signs, gross and microscopic findings were evaluated, viral nucleic acid detection was assessed by in situ hybridization (ISH) in several tissues and viral RNA was detected by RT-PCR in trachea, kidney and nasal and cloacal swabs. Italy 02 serotype was demonstrated to cause severe respiratory and renal damage in one-day old chicks but not in adult hens in which only respiratory disease and drop in egg production was observed. The use of ISH technique demonstrated the presence of viral RNA in nasal turbinates prior to trachea, but more consistent and longer replication periods in enterocytes of lower gastrointestinal tract. The detection of viral nucleic acid in gut by RT-PCR was consistent and more persistent viral shedding was detected in faeces than in nasal exudates. We describe a complete update of IBV distribution in tissues by the use of molecular techniques and we also provide and in-depth pathological characterization of the new Italy 02 IBV serotype. Furthermore, new data about IBV pathogenesis essential in field control is afforded. Copyright © 2011 Elsevier B.V. All rights reserved.

Adeno-Associated Virus–Mediated Gene Therapy for Metabolic Myopathy

PubMed Central

Mah, Cathryn S.; Soustek, Meghan S.; Todd, A. Gary; McCall, Angela; Smith, Barbara K.; Corti, Manuela; Falk, Darin J.

2013-01-01

Abstract Metabolic myopathies are a diverse group of rare diseases in which impaired breakdown of stored energy leads to profound muscle dysfunction ranging from exercise intolerance to severe muscle wasting. Metabolic myopathies are largely caused by functional deficiency of a single gene and are generally subcategorized into three major types of metabolic disease: mitochondrial, lipid, or glycogen. Treatment varies greatly depending on the biochemical nature of the disease, and unfortunately no definitive treatments exist for metabolic myopathy. Since this group of diseases is inherited, gene therapy is being explored as an approach to personalized medical treatment. Adeno-associated virus–based vectors in particular have shown to be promising in the treatment of several forms of metabolic myopathy. This review will discuss the most recent advances in gene therapy efforts for the treatment of metabolic myopathies. PMID:24164240

Viral-associated lymphoid proliferations☆

PubMed Central

Pittaluga, Stefania

2013-01-01

The histological spectrum of viral-associated lymphoid proliferations is quite broad, ranging from reactive lymphadenitis to atypical proliferations mimicking classical Hodgkin lymphoma or non-Hodgkin lymphoma. Virally associated reactive lesions can appear quite alarming on histological examination, because of direct (cytopathic) and indirect viral-induced changes eliciting a polymorphic cellular host response. In addition, the atypical lymphoid proliferation may show aberrant phenotypic features as well as restricted/clonal gene immunoglobulin or T-cell receptor rearrangements, further complicating the interpretation. In order to achieve an accurate diagnosis, it is important to be aware of the clinical history, including family history and ethnic background, clinical presentation, symptoms, and extent of the disease. Among the clinical data, particular emphasis should be placed on serology and viral load studies, and the use of immunosuppressive drugs. The clinical course and outcome vary greatly, from an indolent, self-limited to aggressive clinical course, blurring at times the distinction between neoplastic and reactive proliferations. It is now recognized that immunosenescence also plays a significant role in the development of these viral-associated lymphoid proliferations, and new entities have been described in recent years. In this review we discuss mostly Epstein–Barr virus-associated viral proliferations that may be confused with lymphomas, which the practicing pathologist may encounter. PMID:23537914

Capsular serotypes, virulence-associated genes and antimicrobial susceptibility of Streptococcus suis isolates from pigs in Korea.

PubMed

Oh, Sang-Ik; Jeon, Albert Byungyun; Jung, Byeong-Yeal; Byun, Jae-Won; Gottschalk, Marcelo; Kim, Aeran; Kim, Jong Wan; Kim, Ha-Young

2017-04-20

Streptococcus suis is an important pig pathogen with potential for human transmission. The serotype distributions and phenotypic characteristics vary over time and among regions; however, little is known about the characteristics of S. suis isolates in Korea. In this study, 240 S. suis isolates collected from pigs in Korea in 2009-2010 were serotyped by coagglutination tests, subsequently screened for three virulence-associated genes (mrp, epf and sly) and tested for antimicrobial susceptibility. As for 80 isolates, the serotypes of which were relevant to human infections, clonal complexes (CCs) were further identified by PCR. Serotype 3 was the most prevalent (15.8%), followed by serotype 2 (15.0%), with geographical variation for each serotype. Overall, 55.4% of the isolates carried mrp, whereas only 3.8% carried epf. CC25 was the most prevalent (41.3%) and was related to serotypes 2 and 9. The isolates showed higher susceptibility to ampicillin (93.4%) and ceftiofur (90.8%) than to the other antimicrobial agents tested. The highest resistance rate was observed to tetracycline (98.0%), followed by erythromycin (88.8%). In addition, the resistance to certain antimicrobials was significantly associated, in part, with virulence-associated genes or serotypes. Therefore, continuous characterization of S. suis is essential for the benefit of veterinary and human medicine.

Long-term functional adeno-associated virus-microdystrophin expression in the dystrophic CXMDj dog.

PubMed

Koo, Taeyoung; Okada, Takashi; Athanasopoulos, Takis; Foster, Helen; Takeda, Shin'ichi; Dickson, George

2011-09-01

Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+)  T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD. Copyright © 2011 John Wiley & Sons, Ltd.

Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients.

PubMed

Leborgne, Christian; Latournerie, Virginie; Boutin, Sylvie; Desgue, Diana; Quéré, Aliénor; Pignot, Elodie; Collaud, Fanny; Charles, Séverine; Simon Sola, Marcelo; Masat, Elisa; Jouen, Fabienne; Boyer, Olivier; Masurier, Carole; Mingozzi, Federico; Veron, Philippe

2018-03-16

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans. Copyright © 2018. Published by Elsevier Inc.

Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales

PubMed Central

Negrete, Alejandro; Kotin, Robert M.

2007-01-01

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g.≥1016 particles per production run, utilizes Baculovirus Expression Vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred-tank bioreactors are single-use, disposable bioreactors, e.g. Wave™. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave™ bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave™ bioreactors. Comparable yields of rAAV, ~2e+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration. Keywords: adeno-associated vectors; large-scale production; stirred tank bioreactor; wave bioreactor; gene therapy. PMID:17606302

Using Rapid Diagnostic Tests as a Source of Viral RNA for Dengue Serotyping by RT-PCR - A Novel Epidemiological Tool

PubMed Central

Vongsouvath, Manivanh; Phommasone, Koukeo; Sengvilaipaseuth, Onanong; Kosoltanapiwat, Nathamon; Chantratita, Narisara; Blacksell, Stuart D.; Lee, Sue J.; de Lamballerie, Xavier; Mayxay, Mayfong; Keomany, Sommay; Newton, Paul N.; Dubot-Pérès, Audrey

2016-01-01

Background Dengue virus infection causes major public health problems in tropical and subtropical areas. In many endemic areas, including the Lao PDR, inadequate access to laboratory facilities is a major obstacle to surveillance and study of dengue epidemiology. Filter paper is widely used for blood collection for subsequent laboratory testing for antibody and nucleic acid detection. For the first time, we demonstrate that dengue viral RNA can be extracted from dengue rapid diagnostic tests (RDT) and then submitted to real-time RT-PCR for serotyping. Methodology/Principal Findings We evaluated the Standard Diagnostics (SD) Bioline Dengue Duo RDT, a commonly used test in dengue endemic areas. First, using the QIAamp RNA kit, dengue RNA was purified from the sample pad of the NS1 RDT loaded with virus isolates of the four serotypes, then quantified by RT-PCR. We observed greater recovery of virus, with a mean of 27 times more RNA recovered from RDT, than from filter paper. Second, we evaluated dengue NS1 RDTs from patients at Mahosot Hospital, Vientiane, (99 patients) and from rural Salavan Provincial Hospital (362 patients). There was good agreement between dengue RT-PCR from NS1 RDT with RT-PCR performed on RNA extracted from patient sera, either using RDT loaded with blood (82.8% and 91.4%, in Vientiane and Salavan, respectively) or serum (91.9% and 93.9%). There was 100% concordance between RDT and serum RT-PCR of infecting dengue serotype. Conclusions/Significance Therefore, the collection of NS1 positive RDTs, which do not require cold storage, may be a novel approach for dengue serotyping by RT-PCR and offers promising prospects for the collection of epidemiological data from previously inaccessible tropical areas to aid surveillance and public health interventions. PMID:27159058

Increasing airline travel may facilitate co-circulation of multiple dengue virus serotypes in Asia.

PubMed

Tian, Huaiyu; Sun, Zhe; Faria, Nuno Rodrigues; Yang, Jing; Cazelles, Bernard; Huang, Shanqian; Xu, Bo; Yang, Qiqi; Pybus, Oliver G; Xu, Bing

2017-08-01

The incidence of dengue has grown dramatically in recent decades worldwide, especially in Southeast Asia and the Americas with substantial transmission in 2014-2015. Yet the mechanisms underlying the spatio-temporal circulation of dengue virus (DENV) serotypes at large geographical scales remain elusive. Here we investigate the co-circulation in Asia of DENV serotypes 1-3 from 1956 to 2015, using a statistical framework that jointly estimates migration history and quantifies potential predictors of viral spatial diffusion, including socio-economic, air transportation and maritime mobility data. We find that the spread of DENV-1, -2 and -3 lineages in Asia is significantly associated with air traffic. Our analyses suggest the network centrality of air traffic hubs such as Thailand and India contribute to seeding dengue epidemics, whilst China, Cambodia, Indonesia, and Singapore may establish viral diffusion links with multiple countries in Asia. Phylogeographic reconstructions help to explain how growing air transportation networks could influence the dynamics of DENV circulation.

Increasing airline travel may facilitate co-circulation of multiple dengue virus serotypes in Asia

PubMed Central

Sun, Zhe; Faria, Nuno Rodrigues; Yang, Jing; Cazelles, Bernard; Huang, Shanqian; Xu, Bo; Yang, Qiqi; Pybus, Oliver G.; Xu, Bing

2017-01-01

The incidence of dengue has grown dramatically in recent decades worldwide, especially in Southeast Asia and the Americas with substantial transmission in 2014–2015. Yet the mechanisms underlying the spatio-temporal circulation of dengue virus (DENV) serotypes at large geographical scales remain elusive. Here we investigate the co-circulation in Asia of DENV serotypes 1–3 from 1956 to 2015, using a statistical framework that jointly estimates migration history and quantifies potential predictors of viral spatial diffusion, including socio-economic, air transportation and maritime mobility data. We find that the spread of DENV-1, -2 and -3 lineages in Asia is significantly associated with air traffic. Our analyses suggest the network centrality of air traffic hubs such as Thailand and India contribute to seeding dengue epidemics, whilst China, Cambodia, Indonesia, and Singapore may establish viral diffusion links with multiple countries in Asia. Phylogeographic reconstructions help to explain how growing air transportation networks could influence the dynamics of DENV circulation. PMID:28771468

Capsular serotypes, virulence-associated genes and antimicrobial susceptibility of Streptococcus suis isolates from pigs in Korea

PubMed Central

OH, Sang-Ik; JEON, Albert Byungyun; JUNG, Byeong-Yeal; BYUN, Jae-Won; GOTTSCHALK, Marcelo; KIM, Aeran; KIM, Jong Wan; KIM, Ha-Young

2017-01-01

Streptococcus suis is an important pig pathogen with potential for human transmission. The serotype distributions and phenotypic characteristics vary over time and among regions; however, little is known about the characteristics of S. suis isolates in Korea. In this study, 240 S. suis isolates collected from pigs in Korea in 2009–2010 were serotyped by coagglutination tests, subsequently screened for three virulence-associated genes (mrp, epf and sly) and tested for antimicrobial susceptibility. As for 80 isolates, the serotypes of which were relevant to human infections, clonal complexes (CCs) were further identified by PCR. Serotype 3 was the most prevalent (15.8%), followed by serotype 2 (15.0%), with geographical variation for each serotype. Overall, 55.4% of the isolates carried mrp, whereas only 3.8% carried epf. CC25 was the most prevalent (41.3%) and was related to serotypes 2 and 9. The isolates showed higher susceptibility to ampicillin (93.4%) and ceftiofur (90.8%) than to the other antimicrobial agents tested. The highest resistance rate was observed to tetracycline (98.0%), followed by erythromycin (88.8%). In addition, the resistance to certain antimicrobials was significantly associated, in part, with virulence-associated genes or serotypes. Therefore, continuous characterization of S. suis is essential for the benefit of veterinary and human medicine. PMID:28250312

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

PubMed Central

Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

2016-01-01

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

PubMed

Li, Hua; Zhang, Feng-Lan; Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

2015-01-01

The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

Viral Vector-Based Targeting of miR-21 in Cardiac Nonmyocyte Cells Reduces Pathologic Remodeling of the Heart

PubMed Central

Ramanujam, Deepak; Sassi, Yassine; Laggerbauer, Bernhard; Engelhardt, Stefan

2016-01-01

Systemic inhibition of miR-21 has proven effective against myocardial fibrosis and dysfunction, while studies in cardiac myocytes suggested a protective role in this cell type. Considering potential implications for therapy, we aimed to determine the cell fraction where miR-21 exerts its pathological activity. We developed a viral vector-based strategy for gene targeting of nonmyocyte cardiac cells in vivo and compared global to cardiac myocyte-specific and nonmyocyte-specific deletion of miR-21 in chronic left ventricular pressure overload. Murine moloney virus and serotype 9 of adeno-associated virus were engineered to encode improved Cre recombinase for genetic deletion in miR-21fl/fl mice. Pericardial injection of murine moloney virus-improved Cre recombinase to neonates achieved highly selective genetic ablation of miR-21 in nonmyocyte cardiac cells, identified as cardiac fibroblasts and endothelial cells. Upon left ventricular pressure overload, cardiac function was only preserved in mice with miR-21 deficiency in nonmyocyte cardiac cells, but not in mice with global or cardiac myocyte-specific ablation. Our data demonstrate that miR-21 exerts its pathologic activity directly in cardiac nonmyocytes and encourage further development of antimiR-21 therapy toward cellular tropism. PMID:27545313

A 1-Year Quantitative Survey of Noro-, Adeno-, Human Boca-, and Hepatitis E Viruses in Raw and Secondarily Treated Sewage from Two Plants in Norway.

PubMed

Myrmel, M; Lange, H; Rimstad, E

2015-09-01

A study of enteric viruses in raw and treated sewage from two secondary treatment plants, which received sewage from Oslo city (plant A) and small municipalities in Hedmark county in Norway (plant B), showed high levels of noro-, adeno-, and bocavirus throughout the year. A seasonal variation was observed for adeno- and GII norovirus with higher levels during winter and bocavirus that had more positive samples during winter. The virus concentrations in raw sewage were comparable in the two plants, with medians (log10 genome copies per liter) of 6.1, 6.3, 6.0, and 4.5 for noro GI, noro GII, adeno-, and bocavirus, respectively. The level of hepatitis E virus was not determined as it was below the limit of quantification. The mean log10 virus reduction was 0.55 (plant A) and 1.44 (plant B) with the highest reduction found in the plant with longer hydraulic retention time. The adenoviruses were dominantly serotype 41, while serotype 12 appeared sporadically. Of the 102 raw and treated sewage samples that were tested, eight were positive for hepatitis E virus of which four were from treated sewage. Two of the four obtained gene sequences from hepatitis E virus originated from the rural sewage samples and showed high similarity with a genotype 3 strain of hepatitis E virus detected in local piglets. Two other hepatitis E virus sequences obtained from urban sewage samples showed high similarities with genotype 3 strains isolated from urban sewage in Spain and a human genotype 1 isolate from India. The study gives information on the levels of noroviruses in raw and treated sewage, which is valuable to risk assessment, information indicating that some infections with hepatitis E viruses in Norway have a regional origin and that human bocavirus 2 and 3 are prevalent in the Norwegian population.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

PubMed

Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

1996-07-15

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material

PubMed Central

Lock, Martin; McGorray, Susan; Auricchio, Alberto; Ayuso, Eduard; Beecham, E. Jeffrey; Blouin-Tavel, Véronique; Bosch, Fatima; Bose, Mahuya; Byrne, Barry J.; Caton, Tina; Chiorini, John A.; Chtarto, Abdelwahed; Clark, K. Reed; Conlon, Thomas; Darmon, Christophe; Doria, Monica; Douar, Anne; Flotte, Terence R.; Francis, Joyce D.; Francois, Achille; Giacca, Mauro; Korn, Michael T.; Korytov, Irina; Leon, Xavier; Leuchs, Barbara; Lux, Gabriele; Melas, Catherine; Mizukami, Hiroaki; Moullier, Philippe; Müller, Marcus; Ozawa, Keiya; Philipsberg, Tina; Poulard, Karine; Raupp, Christina; Rivière, Christel; Roosendaal, Sigrid D.; Samulski, R. Jude; Soltys, Steven M.; Surosky, Richard; Tenenbaum, Liliane; Thomas, Darby L.; van Montfort, Bart; Veres, Gabor; Wright, J. Fraser; Xu, Yili; Zelenaia, Olga; Zentilin, Lorena

2010-01-01

Abstract A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus–free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 × 1011 particles/ml; 95% confidence interval [CI], 7.89 × 1011 to 1.05 × 1012 particles/ml), vector genomes ({X}, 3.28 × 1010 vector genomes/ml; 95% CI, 2.70 × 1010 to 4.75 × 1010 vector genomes/ml), transducing units ({X}, 5.09 × 108 transducing units/ml; 95% CI, 2.00 × 108 to 9.60 × 108 transducing units/ml), and infectious units ({X}, 4.37 × 109 TCID50 IU/ml; 95% CI, 2.06 × 109 to 9.26 × 109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited

African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever.

PubMed

Burmakina, G; Malogolovkin, A; Tulman, E R; Zsak, L; Delhon, G; Diel, D G; Shobogorov, N M; Morgunov, Yu P; Morgunov, S Yu; Kutish, G F; Kolbasov, D; Rock, D L

2016-07-01

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.

Prophylaxis and Treatment of Alzheimer's Disease by Delivery of an Adeno-Associated Virus Encoding a Monoclonal Antibody Targeting the Amyloid Beta Protein

PubMed Central

Shimada, Masaru; Abe, Shinya; Takahashi, Toru; Shiozaki, Kazumasa; Okuda, Mitsue; Mizukami, Hiroaki; Klinman, Dennis M.; Ozawa, Keiya; Okuda, Kenji

2013-01-01

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (Aß) protein. Repeated injection of that mAb reduced the accumulation of Aß protein in the brain of human Aß transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0×1010 viral genome of these AAV vectors into C57BL/6 mice generated serum anti-Aß Ab levels up to 0.3 mg/ml. Anti-Aß Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on Aß levels in vivo was examined. A significant decrease in Aß levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-Aß Ab for the prevention and treatment of Alzheimer's disease. PMID:23555563

Current status of non-viral gene therapy for CNS disorders

PubMed Central

Jayant, Rahul Dev; Sosa, Daniela; Kaushik, Ajeet; Atluri, Venkata; Vashist, Arti; Tomitaka, Asahi; Nair, Madhavan

2017-01-01

Introduction Viral and non-viral vectors have been used as methods of delivery in gene therapy for many CNS diseases. Currently, viral vectors such as adeno-associated viruses (AAV), retroviruses, lentiviruses, adenoviruses and herpes simplex viruses (HHV) are being used as successful vectors in gene therapy at clinical trial levels. However, many disadvantages have risen from their usage. Non-viral vectors like cationic polymers, cationic lipids, engineered polymers, nanoparticles, and naked DNA offer a much safer option and can therefore be explored for therapeutic purposes. Areas covered This review discusses different types of viral and non-viral vectors for gene therapy and explores clinical trials for CNS diseases that have used these types of vectors for gene delivery. Highlights include non-viral gene delivery and its challenges, possible strategies to improve transfection, regulatory issues concerning vector usage, and future prospects for clinical applications. Expert opinion Transfection efficiency of cationic lipids and polymers can be improved through manipulation of molecules used. Efficacy of cationic lipids is dependent on cationic charge, saturation levels, and stability of linkers. Factors determining efficacy of cationic polymers are total charge density, molecular weights, and complexity of molecule. All of the above mentioned parameters must be taken care for efficient gene delivery. PMID:27249310

Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology

PubMed Central

Sizemore, Rachel J.; Seeger-Armbruster, Sonja; Hughes, Stephanie M.

2016-01-01

Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients. PMID:26888111

Development of a rapid serotyping method for Salmonella enterica using serotype-specific single-nucleotide polymorphisms

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subsp. enterica serotype Enteriditis (S. Enteriditis) is the leading cause of salmonellosis worldwide, including the USA. Many S. enterica serotypes known to cause foodborne disease are associated with broiler meat contamination. While some serotypes are specific to birds (S. e...

Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

PubMed

Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

2015-10-15

The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.

PubMed

Khanam, Saima; Rajendra, Pilankatta; Khanna, Navin; Swaminathan, Sathyamangalam

2007-02-15

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective

Enterovirus serotypes in patients with central nervous system and respiratory infections in Viet Nam 1997-2010.

PubMed

B'Krong, Nguyen Thi Thuy Chinh; Minh, Ngo Ngoc Quang; Qui, Phan Tu; Chau, Tran Thi Hong; Nghia, Ho Dang Trung; Do, Lien Anh Ha; Nhung, Nguyen Ngoc; Van Vinh Chau, Nguyen; Thwaites, Guy; Van Tan, Le; van Doorn, H Rogier; Thanh, Tran Tan

2018-04-12

Enteroviruses are the most common causative agents of human illness. Enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (Enterovirus A71 and D68), and are of interest as emerging viruses. Here, we typed Enterovirus A-D (EV) from central nervous system (CNS) and respiratory infections in Viet Nam. Data and specimens from prospective observational clinical studies conducted between 1997 and 2010 were used. Species and serotypes were determined using type-specific RT-PCR and viral protein 1 or 4 (VP1, VP4) sequencing. Samples from patients with CNS infection (51 children - 10 CSF and 41 respiratory/rectal swabs) and 28 adults (28 CSF) and respiratory infection (124 children - 124 respiratory swabs) were analysed. Twenty-six different serotypes of the four Enterovirus species (A-D) were identified, including EV-A71 and EV-D68. Enterovirus B was associated with viral meningitis in children and adults. Hand, foot and mouth disease associated Enteroviruses A (EV-A71 and Coxsackievirus [CV] A10) were detected in children with encephalitis. Diverse serotypes of all four Enterovirus species were found in respiratory samples, including 2 polio-vaccine viruses, but also 8 CV-A24 and 8 EV-D68. With the exception of EV-D68, the relevance of these viruses in respiratory infection remains unknown. We describe the diverse spectrum of enteroviruses from patients with CNS and respiratory infections in Viet Nam between 1997 and 2010. These data confirm the global circulation of Enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response.

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Weihong; Wu Jianqing; Zhong Li

2006-09-30

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency ofmore » conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of

An infant with concurrent serotype 6C invasive pneumococcal disease and infectious mononucleosis.

PubMed

Nishikawa-Nakamura, Naoko; Okada, Takafumi; Nishimura, Keiko; Iwai, Tsuyako; Ubukata, Kimiko; Iwata, Satoshi; Iwai, Asayuki

2017-11-01

Streptococcus pneumoniae is a main causative agent of serious invasive bacterial infections. However, concurrent infection with invasive pneumococcal disease (IPD) and viral infectious mononucleosis (IM) is rare. We report an infant with serotype 6C infection causing IPD occurring simultaneously with IM. A previously healthy 11-month-old girl referred to our hospital because of fever, leukopenia, and elevated C-reactive protein presented to us with disturbance of consciousness, tachycardia, tachypnea and agranulocytosis. Other findings included tonsillitis with purulent exudates and white spots, bilateral cervical adenopathy, and hepatosplenomegaly. We diagnosed her illness as sepsis and administered a broad-spectrum antibiotic, an antiviral agent, and granulocyte transfusions. After treatment was initiated, fever gradually decreased and general condition improved. IPD was diagnosed based upon isolation of S. pneumoniae of serotype 6C from blood cultures obtained on admission. Concurrently the girl had IM, based upon quantitation of Epstein-Barr viral DNA copies in blood and fluctuating serum antibody titers. Although simultaneous IPD and IM is a rare occurrence, this possibility is important to keep in mind. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

PubMed

Iskarpatyoti, Jason A; Morse, E Ashley; McClung, R Paul; Ikizler, Miné; Wetzel, J Denise; Contractor, Nikhat; Dermody, Terence S

2012-11-25

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk. Copyright © 2012 Elsevier Inc. All rights reserved.

Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology.

PubMed

Sizemore, Rachel J; Seeger-Armbruster, Sonja; Hughes, Stephanie M; Parr-Brownlie, Louise C

2016-04-01

Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients. Copyright © 2016 the American Physiological Society.

NLX-P101, an adeno-associated virus gene therapy encoding glutamic acid decarboxylase, for the potential treatment of Parkinson's disease.

PubMed

Diaz-Nido, Javier

2010-07-01

Parkinson's disease (PD) is a neurodegenerative disease affecting nigrostriatal dopaminergic neurons. Dopamine depletion in the striatum leads to functional changes in several deep brain nuclei, including the subthalamic nucleus (STN), which becomes disinhibited and perturbs the control of body movement. Although there is no cure for PD, some pharmacological and surgical treatments can significantly improve the functional ability of patients, particularly in the early stages of the disease. Among neurodegenerative diseases, PD is a particularly suitable target for gene therapy because the neuropathology is largely confined to a relatively small region of the brain. Neurologix Inc is developing NLX-P101 (AAV2-GAD), an adeno-associated viral vector encoding glutamic acid decarboxylase (GAD), for the potential therapy of PD. As GAD potentiates inhibitory neurotransmission from the STN, sustained expression of GAD in the STN by direct delivery of NLX-P101 decreases STN overactivation. This procedure was demonstrated to be a safe and efficient method of reducing motor deficits in animal models of PD. A phase I clinical trial has demonstrated that NLX-P101 was safe and indicated the efficacy of this approach in patients with PD. Results from an ongoing phase II clinical trial of NLX-P101 are awaited to establish the clinical efficacy of this gene therapy.

Molecular typing of enteroviruses associated with viral meningitis in Cyprus, 2000-2002.

PubMed

Richter, Jan; Koptides, Dana; Tryfonos, Christina; Christodoulou, Christina

2006-08-01

Human enteroviruses are responsible for a wide spectrum of clinical diseases affecting many different organ systems. Although infection is usually asymptomatic, infections of the central nervous system manifested as meningitis or encephalitis can pose a serious public health problem, especially during outbreaks. In this study, samples from 218 patients diagnosed with enteroviral meningitis between January 2000 and December 2002 were analysed in order to assess the epidemiology of human enteroviruses as a cause of viral meningitis in Cyprus. A new typing strategy, based on partial sequencing of the 5' non-coding region (5'NCR), prediction of type, and selection of type-specific primers for sensitive VP1 PCR amplification, was developed. As clustering in the 5'NCR was concordant with clustering in the VP1 region, quick and reliable typing by VP1 sequencing was achieved without virus isolation in cell culture. The most frequent enterovirus serotypes identified were Human echovirus 30 (55.5%), Human echovirus 13 (15.1%), Human echovirus 6 (13.8%) and Human echovirus 9 (8.3%). Human coxsackieviruses B2, B1 and B5, Human echovirus 4, Human enterovirus 71 and Human coxsackievirus A6 represented rather rare serotypes. This is the first molecular epidemiological study of enterovirus meningitis in Cyprus. Serotype distribution corresponded basically with observations in other European countries, suggesting the spread of enteroviruses by tourism.

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

PubMed

Barbash, I M; Cecchini, S; Faranesh, A Z; Virag, T; Li, L; Yang, Y; Hoyt, R F; Kornegay, J N; Bogan, J R; Garcia, L; Lederman, R J; Kotin, R M

2013-03-01

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Toward exascale production of recombinant adeno-associated virus for gene transfer applications.

PubMed

Cecchini, S; Negrete, A; Kotin, R M

2008-06-01

To gain acceptance as a medical treatment, adeno-associated virus (AAV) vectors require a scalable and economical production method. Recent developments indicate that recombinant AAV (rAAV) production in insect cells is compatible with current good manufacturing practice production on an industrial scale. This platform can fully support development of rAAV therapeutics from tissue culture to small animal models, to large animal models, to toxicology studies, to Phase I clinical trials and beyond. Efforts to characterize, optimize and develop insect cell-based rAAV production have culminated in successful bioreactor-scale production of rAAV, with total yields potentially capable of approaching the exa-(10(18)) scale. These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases such as Duchenne muscular dystrophy, for which large amounts of recombinant vector are essential for successful outcome.

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay.

PubMed

Lebani, Kebaneilwe; Jones, Martina L; Watterson, Daniel; Ranzoni, Andrea; Traves, Renee J; Young, Paul R; Mahler, Stephen M

2017-01-01

The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst

Co-circulating serotypes in a dengue fever outbreak: Differential hematological profiles and phylogenetic relationships among viruses.

PubMed

Carmo, Andreia Moreira Dos Santos; Suzuki, Rodrigo Buzinaro; Cabral, Aline Diniz; Costa, Renata Torres da; Massari, Gabriela Pena; Riquena, Michele Marcondes; Fracasso, Helio Augusto Alves; Eterovic, Andre; Marcili, Arlei; Sperança, Márcia Aparecida

2017-05-01

Dengue virus, represented by four distinct, genetically diverse serotypes, is the etiologic agent of asymptomatic to severe hemorrhagic diseases. The spatiotemporal dynamics of dengue serotypes and its association to specific diseases vary among the different regions worldwide. By 2007, and in São Paulo State, Brazil, dengue-case concentration in urban centers had changed to increased incidence in small- and medium-sized towns, the case of Marília. The aim of this article was to distinguish dengue serotypes circulating during the 2007 Marília outbreak and define their association to demographic and hematological patient profiles, as well as the phylogenetic relationships among the different viruses. PCR amplicons corresponding to the junction of capsid and dengue pre-membrane encoding genes, obtained from dengue serologically positive patients, were sequenced. Hematological and demographic data of patients with different Dengue serotypes were evaluated by univariate and bivariate statistics. Dengue PCR sequences were used in phylogenetic relationships analyzed for maximum parsimony. Molecular typing confirmed co-circulation of the dengue serotypes 1 (DENV1) and 3 (DENV3), which presented divergent correlation patterns with regard to hematological descriptors. The increase in atypical lymphocytes, a likely indication of virus load, could be significantly associated to a decrease in leukocyte counts in the DENV3 group and platelet in the DENV1. Phylogenetic reconstitution revealed the introduction of DENV1 from northern Brazil and local divergence of DENV3 by either microevolution or viral introduction from other geographical regions or both. Dengue dynamics showed regional molecular-epidemiologic specificity, which has important implications for introduction of vaccines, disease management, and transmission control. Copyright © 2017 Elsevier B.V. All rights reserved.

Pseudotyped adeno-associated virus 2/9-delivered CCL11 shRNA alleviates lung inflammation in an allergen-sensitized mouse model.

PubMed

Wu, Chia-Jen; Huang, Wen-Chung; Chen, Li-Chen; Shen, Chia-Rui; Kuo, Ming-Ling

2012-11-01

Airway infiltration by eosinophils is a major characteristic of chronic asthma. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as the major chemokine for eosinophil recruitment. Pseudotyped adeno-associated virus (AAV) 2/9, composed by the AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether pseudotyped AAV2/9 virus carrying the small hairpin RNA targeting CCL11 and expressed by CMV/U6 promoter could reduce eosinophilia and asthmatic responses in mite allergen-sensitized mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 allergen (rDp2). AAV2/9 viral vectors were intratracheally injected three days before the first challenge. AAV2/9 sh47 virus significantly reduced airway hyperresponsiveness, airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-10, were also significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. The results suggest that AAV2/9 sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the CMV/U6 promoter with AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma.

Pseudotyped Adeno-Associated Virus 2/9-Delivered CCL11 shRNA Alleviates Lung Inflammation in an Allergen-Sensitized Mouse Model

PubMed Central

Wu, Chia-Jen; Huang, Wen-Chung; Chen, Li-Chen; Shen, Chia-Rui

2012-01-01

Abstract Airway infiltration by eosinophils is a major characteristic of chronic asthma. CCL11 (eotaxin-1) is secreted by lung epithelial cells and functions as the major chemokine for eosinophil recruitment. Pseudotyped adeno-associated virus (AAV) 2/9, composed by the AAV2 rep and AAV9 cap genes, can efficiently target lung epithelial cells and might carry gene sequences with therapeutic potential for asthma. This study aimed to determine whether pseudotyped AAV2/9 virus carrying the small hairpin RNA targeting CCL11 and expressed by CMV/U6 promoter could reduce eosinophilia and asthmatic responses in mite allergen-sensitized mice. Mice were sensitized by intraperitoneal and challenged by intratracheal injection with recombinant Dermatophagoides pteronyssinus group 2 allergen (rDp2). AAV2/9 viral vectors were intratracheally injected three days before the first challenge. AAV2/9 sh47 virus significantly reduced airway hyperresponsiveness, airway resistance, CCL11 levels, and eosinophilia in the lungs of sensitized mice. Th2 cytokines, including interleukins (IL)-4, IL-5, and IL-10, were also significantly reduced in the bronchoalveolar lavage fluid of AAV2/9 sh47 virus-treated mice. Th2 cytokine levels were also reduced in rDp2-stimulated mediastinal lymphocytes in treated mice. However, serum levels of rDp2-specific IgG1 and IgE, as well as Th2 cytokine levels in rDp2-stimulated splenocyte culture supernatants, were comparable to the sensitized control group. The results suggest that AAV2/9 sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the CMV/U6 promoter with AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma. PMID:22913580

[Identification of rotavirus associated to serotype G2 in Yucatan, Mexico].

PubMed

Gonzales-Loza, M del R; Polanco-Marín, G G; Puerto-Solis, M

2000-01-01

In the present study, rotavirus G2 serotype was identified from fecal samples of children with gastroenteritis from the city of Merida, Yucatan, Mexico. Virological diagnosis of disease was performed using polycrylamide gel electrophoresis and immunoenzymatic assay. Out of 149 analyzed samples 25 (16.7%) gave positive reaction to rotavirus groups A, of these 23 (92%) were identified as serotype g2, subgroup i and electrophoretic short pattern, whereas 2 (8%) were identified as subgroups II and electrophoretic long pattern, however, the G serotype was not possible to determine. Rotavirus G serotype has not been detected in more than 90% of samples since 1985. This indicates that the number of people susceptible to G2 serotype within the population has increased over recent years, which perhaps indicates that an important outbreak of acute infectious diarrhea caused by the rotavirus G2 serotype may be forthcoming.

Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

PubMed Central

Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

2016-01-01

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal

Adeno-associated virus inverted terminal repeats stimulate gene editing.

PubMed

Hirsch, M L

2015-02-01

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

Population-based genetic epidemiologic analysis of Chlamydia trachomatis serotypes and lack of association between ompA polymorphisms and clinical phenotypes.

PubMed

Millman, Kim; Black, Carolyn M; Stamm, Walter E; Jones, Robert B; Hook, Edward W; Martin, David H; Bolan, Gail; Tavaré, Simon; Dean, Deborah

2006-03-01

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted diseases worldwide. Urogenital strains are classified into serotypes and genotypes based on the major outer membrane protein and its gene, ompA, respectively. Studies of the association of serotypes with clinical signs and symptoms have produced conflicting results while no studies have evaluated associations with ompA polymorphisms. We designed a population-based cross-sectional study of 344 men and women with urogenital chlamydial infections (excluding co-pathogen infections) presenting to clinics serving five U.S. cities from 1995 to 1997. Signs, symptoms and sequelae of chlamydial infection (mucopurulent cervicitis, vaginal or urethral discharge; dysuria; lower abdominal pain; abnormal vaginal bleeding; and pelvic inflammatory disease) were analyzed for associations with serotype and ompA polymorphisms. One hundred and fifty-three (44.5%) of 344 patients had symptoms consistent with urogenital chlamydial infection. Gender, reason for visit and city were significant independent predictors of symptom status. Men were 2.2 times more likely than women to report any symptoms (P=0.03) and 2.8 times more likely to report a urethral discharge than women were to report a vaginal discharge in adjusted analyses (P=0.007). Differences in serotype or ompA were not predictive except for an association between serotype F and pelvic inflammatory disease (P=0.046); however, the number of these cases was small. While there was no clinically prognostic value associated with serotype or ompA polymorphism for urogenital chlamydial infections except for serotype F, future studies might utilize multilocus genomic typing to identify chlamydial strains associated with clinical phenotypes.

Intracranial delivery of Interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway

PubMed Central

Yang, Junling; Kou, Jinghong; Lim, Jeong-Eun; Lalonde, Robert; Fukuchi, Ken-ichiro

2015-01-01

Interleukin-17A (IL-17A) is generally considered as one of the pathogenic factors involved in multiple sclerosis (MS). Indirect evidence for this is that IL-17A-producing T helper 17 (Th17) cells preferentially accumulate in lesions of MS and experimental autoimmune encephalomyelitis (EAE). However, a direct involvement of IL-17A in MS pathogenesis is still an open question. In this study, we overexpressed IL-17A in the brains of mice (IL-17A-in-Brain mice) via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated gene delivery. In spite of high levels of IL-17A expression in the brain and blood, IL-17A-in-Brain mice exhibit no inflammatory responses and no abnormalities in motor coordination and spatial orientation. Unexpectedly, IL-17A-in-Brain mice show decreases in body weight and adipose tissue mass and an improvement in glucose tolerance and insulin sensitivity. IL-17A enhances glucose uptake in PC12 cells by activation of AKT. Our results provide direct evidence for the first time that IL-17A overexpression in the central nervous system does not cause physical and learning disabilities and neuroinflammation and suggest that IL-17A may regulate glucose metabolism through the AKT signaling pathway. PMID:26562537

Elastin-like polypeptide matrices for enhancing adeno-associated virus-mediated gene delivery to human neural stem cells.

PubMed

Kim, J-S; Chu, H S; Park, K I; Won, J-I; Jang, J-H

2012-03-01

The successful development of efficient and safe gene delivery vectors continues to be a major obstacle to gene delivery in stem cells. In this study, we have developed an elastin-like polypeptide (ELP)-mediated adeno-associated virus (AAV) delivery system for transducing fibroblasts and human neural stem cells (hNSCs). AAVs have significant promise as therapeutic vectors because of their safety and potential for use in gene targeting in stem cell research. ELP has been recently employed as a biologically inspired 'smart' biomaterial that exhibits an inverse temperature phase transition, thereby demonstrating promise as a novel drug carrier. The ELP that was investigated in this study was composed of a repetitive penta-peptide with [Val-Pro-Gly-Val-Gly]. A novel AAV variant, AAV r3.45, which was previously engineered by directed evolution to enhance transduction in rat NSCs, was nonspecifically immobilized onto ELPs that were adsorbed beforehand on a tissue culture polystyrene surface (TCPS). The presence of different ELP quantities on the TCPS led to variations in surface morphology, roughness and wettability, which were ultimately key factors in the modulation of cellular transduction. Importantly, with substantially reduced viral quantities compared with bolus delivery, ELP-mediated AAV delivery significantly enhanced delivery efficiency in fibroblasts and hNSCs, which have great potential for use in tissue engineering applications and neurodegenerative disorder treatments, respectively. The enhancement of cellular transduction in stem cells, as well as the feasibility of ELPs for utilization in three-dimensional scaffolds, will contribute to the advancement of gene therapy for stem cell research and tissue regenerative medicine.

Viral Vectors for In Vivo Gene Transfer in Parkinson’s disease: Properties and Clinical Grade Production

PubMed Central

Burger, Corinna; Snyder, Richard O.

2009-01-01

Because Parkinson’s disease is a progressive degenerative disorder that is mainly confined to the basal ganglia, gene transfer to deliver therapeutic molecules is an attractive treatment avenue. The present review focuses on direct in vivo gene transfer vectors that have been developed to a degree that they have been successfully used in animal model of Parkinson’s disease. Accordingly, the properties of recombinant adenovirus, recombinant adeno-associated virus, herpes simplex virus, and lentivirus are described and contrasted. In order for viral vectors to be developed into clinical grade reagents, they must be manufactured and tested to precise regulatory standards. Indeed, clinical lots of viral vectors can be produced in compliance with current Good Manufacturing Practices (cGMPs) regulations using industry accepted manufacturing methodologies, manufacturing controls, and quality systems. The viral vector properties themselves combined with physiological product formulations facilitate long-term storage and direct in vivo administration. PMID:17916354

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

PubMed

Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

2012-03-01

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Partial Correction of the CNS Lysosomal Storage Defect in a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis by Neonatal CNS Administration of an Adeno-Associated Virus Serotype rh.10 Vector Expressing the Human CLN3 Gene

PubMed Central

Sondhi, Dolan; Scott, Emma C.; Chen, Alvin; Hackett, Neil R.; Wong, Andrew M.S.; Kubiak, Agnieszka; Nelvagal, Hemanth R.; Pearse, Yewande; Cotman, Susan L.; Cooper, Jonathan D.

2014-01-01

Abstract Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3Δex7/8 knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3Δex7/8 mice were administered 3×1010 genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3Δex7/8 mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3Δex7/8 mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3Δex7/8 mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira

2012-01-06

Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors,more » therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.« less

Population dynamics and antimicrobial resistance of the most prevalent poultry-associated Salmonella serotypes.

PubMed

Shah, Devendra H; Paul, Narayan C; Sischo, Willium C; Crespo, Rocio; Guard, Jean

2017-03-01

Salmonella spp. is the most predominant bacterial cause of foodborne gastroenteritis in humans. Due to the risk of human infection associated with poultry products and the prevalence of antimicrobial resistance, Salmonella also poses a significant challenge to commercial poultry production. During the last decade (2002 to 2012), the 12 most prevalent poultry-associated Salmonella serotypes (MPPSTs) were frequently and consistently isolated from poultry products in the United States. These MPPSTs and their percent prevalence in poultry products include Kentucky (4%), Enteritidis (2%) Heidelberg (2%), Typhimurium (2%), S. I 4,[5],12:i:- (0.31%), Montevideo (0.20%), Infantis (0.16%) Schwarzengrund (0.15%), Hadar (0.15%), Mbandaka (0.13%), Thompson (0.12%), and Senftenberg (0.04%). All MPPSTs except Kentucky are among the top 30 clinically significant serotypes that cause human illnesses in the United States. However with the exception of a few widely studied serotypes such as S. Enteritidis and Typhimurium, the ecology and epidemiology of the majority of MPPSTs still remain poorly investigated. Published data from the United States suggests that MPPSTs such as Heidelberg, Typhimurium, Kentucky, and Sentfenberg are more likely to be multi-drug resistant (MDR, ≥3 antimicobial classes) whereas Enteritidis, Montevideo, Schwarzengrund, Hadar, Infantis, Thompson, and Mbandaka are generally pan-susceptible or display resistance to fewer antimicobials. In contrast, the majority of MPPSTs isolated globally have been reported to display MDR phenotype. There also appears to be an international spread of a few MDR serotypes including Kentucky, Schwarzengrund, Hadar, Thomson, Sentfenberg, and Enteritidis, which may pose significant challenges to the public health. The current knowledge gaps on the ecology, epidemiology, and antimicrobial resistance of MPPSTs are discussed. © 2016 Poultry Science Association Inc.

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

PubMed Central

Korrapati, Anil Babu; Swaminathan, Gokul; Singh, Aarti; Khanna, Navin; Swaminathan, Sathyamangalam

2012-01-01

Background Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. PMID:22848770

Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization.

PubMed

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D

2013-08-02

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization*

PubMed Central

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.

2013-01-01

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669

Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test.

PubMed Central

Dubreuil, J D; Letellier, A; Stenbaek, E; Gottschalk, M

1996-01-01

A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS-PAGE and Western blotting. A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A. pleuropneumoniae, were tested by mixing 25 microL of polystyrene reagent with the same volume of a dense suspension of bacterial cells grown for 18 h. All A. pleuropneumoniae strains had been previously serotyped using standard procedures. The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found. The sensitized polystyrene particles were stable for at least 6 mo. Images Figure 1. PMID:8825998

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency.

PubMed

Mueller, Christian; Gernoux, Gwladys; Gruntman, Alisha M; Borel, Florie; Reeves, Emer P; Calcedo, Roberto; Rouhani, Farshid N; Yachnis, Anthony; Humphries, Margaret; Campbell-Thompson, Martha; Messina, Louis; Chulay, Jeffrey D; Trapnell, Bruce; Wilson, James M; McElvaney, Noel G; Flotte, Terence R

2017-06-07

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors.

PubMed

Gray, Steven J; Foti, Stacey B; Schwartz, Joel W; Bachaboina, Lavanya; Taylor-Blake, Bonnie; Coleman, Jennifer; Ehlers, Michael D; Zylka, Mark J; McCown, Thomas J; Samulski, R Jude

2011-09-01

With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

PubMed

Alves, Sandro; Bode, Julia; Bemelmans, Alexis-Pierre; von Kalle, Christof; Cartier, Nathalie; Tews, Björn

2016-06-20

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

Protein antigen in serotype k Streptococcus mutans clinical isolates.

PubMed

Nakano, K; Nomura, R; Nemoto, H; Lapirattanakul, J; Taniguchi, N; Grönroos, L; Alaluusua, S; Ooshima, T

2008-10-01

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.

Antimicrobial susceptibility, serotypes and genotypes of Pasteurella multocida isolates associated with swine pneumonia in Taiwan.

PubMed

Yeh, Jih-Ching; Lo, Dan-Yuan; Chang, Shao-Kuang; Chou, Chi-Chung; Kuo, Hung-Chih

2017-09-21

Pasteurella multocida (PM) can cause progressive atrophic rhinitis and suppurative bronchopneumonia in pigs. The present study performed antimicrobial susceptibility testing and serotype and genotype identification on the 62 PM strains isolated from the lungs of diseased pigs with respiratory symptoms. Antimicrobial susceptibility testing examined 13 antimicrobial agents (amoxicillin, cefazolin, doxycycline, flumequine, enrofloxacin, florfenicol, kanamycin, lincomycin, Linco-Spectin (lincomycin and spectinomycin), erythromycin, tylosin, tilmicosin and tiamulin). Antimicrobial resistance ratios were over 40% in all of the antimicrobial agents except for cefazolin. The highest levels of resistance (100%) were found for kanamycin, erythromycin and tylosin. The majority of isolated strains was serotype D:L6 (n=35) followed by A:L3 (n=17). Comparison of the antimicrobial resistance levels between the two serotypes showed that the antimicrobial resistance rates were higher in D:L6 than in A:L3 for all the tested antimicrobials except for tylosin and tilmicosin. For PM with erm (B), erm (T) or erm (42), the results showed no significant difference compared with non-resistance gene strains in phenotype. Pulsed-field gel electrophoresis genotyping using Apa I restriction digestion of the genomic DNA demonstrated that there were 17 distinct clusters with a similarity of 85% or more, and the genotyping result was similar to that of serotyping. The results of the present study demonstrated that the PM isolated from diseased pigs in Taiwan was resistant to multiple antimicrobials, and the distribution of antimicrobial resistance was associated with pulsotype and serotype. © British Veterinary Association (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Crystal structure of the SF3 helicase from adeno-associated virus type 2.

PubMed

James, J Anson; Escalante, Carlos R; Yoon-Robarts, Miran; Edwards, Thomas A; Linden, R Michael; Aggarwal, Aneel K

2003-08-01

We report here the crystal structure of an SF3 DNA helicase, Rep40, from adeno-associated virus 2 (AAV2). We show that AAV2 Rep40 is structurally more similar to the AAA(+) class of cellular proteins than to DNA helicases from other superfamilies. The structure delineates the expected Walker A and B motifs, but also reveals an unexpected "arginine finger" that directly implies the requirement of Rep40 oligomerization for ATP hydrolysis and helicase activity. Further, the Rep40 AAA(+) domain is novel in that it is unimodular as opposed to bimodular. Altogether, the structural connection to AAA(+) proteins defines the general architecture of SF3 DNA helicases, a family that includes simian virus 40 (SV40) T antigen, as well as provides a conceptual framework for understanding the role of Rep proteins during AAV DNA replication, packaging, and site-specific integration.

Fowl adenovirus serotype 9 vectored vaccine for protection of avian influenza virus

USDA-ARS?s Scientific Manuscript database

A fowl adenovirus serotype 9, a non-pathogenic large double stranded DNA virus, was developed as a viral vector to express influenza genes as a potential vaccine. Two separate constructs were developed that expressed either the hemagglutinin gene of A/Chicken/Jalisco/2012 (H7) or A/ Chicken/Iowa/20...

Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds

USDA-ARS?s Scientific Manuscript database

Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we se...

[Prevalence of HPV high-risk serotypes detected by PCR in patients with normal cervical cytology at the Hospital Regional Adolfo López Mateos, ISSSTE].

PubMed

Martínez-Portilla, R J; López-Velázquez, J L; Martínez-Rojas, G C; Aguilar-Villagómez, M I; De la Torre-Rendón, F E; Villafán-Bernal, J R

2016-09-01

Is fundamental to determine the prevalence of human papiloma virus (HVP) high-risk serotypes in local and regional population in order for health providers to offer patients, vaccines and treatments against specific population-based serotypes. To determine the prevalence of HPV High risk serotypes detected by PCR in patients with normal cytology from the ISSSTE Adolfo Lopez Mateos Regional Hospital. An observational, descriptive, prospective study was conducted from cervical cytologies and high risk HPV test by PCR in patients from the Regional Hospital Adolfo López Mateos, ISSSTE, during the period January 2013-December 2015. Cases of patients with negative cervical cytology were included. Information about age, the result of cervical cytology and high risk HPV test by PCR was obtained. The overall prevalence of HPV infection and the most prevalent serotypes by age groups were calculated. A total of 3258 cervical smears were performed, of which 2557 were negative (78.4%), from this, the global prevalence of HPV infection was 10.2% (n=262). We found that 1.8% (n = 45) of negative reports had HPV16 infection, 0.5% (n=13) had HPV18 and 8.9% (n = 227) were infected by Viral Pool of other high-risk serotypes. The prevalence of infection by viral pool of high risk serotypes was 11.5% in women <20 years, 12.9% in women between 20-29 years and 22.2% in women between 30-39 years. This prevalence was lower in patients older than 40 years (p<0.05). A higher prevalence of viral pool high risk serotypes was found in patients with normal cytology, than the HPV16 and HPV-8 prevalence, which was significantly higher in women younger than 40 years.

Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

PubMed Central

Li, Linlin; Deng, Xutao; Mee, Edward T.; Collot-Teixeira, Sophie; Anderson, Rob; Schepelmann, Silke; Minor, Philip D.; Delwart, Eric

2014-01-01

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries on the efficiency of viral detection and virus genome coverage were compared. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces. PMID:25497414

ADenoVirus Initiative Study in Epidemiology (ADVISE): Results of a multicentric epidemiology study in Spain.

PubMed

Muñoz-Hernández, A M; Duquesroix, B; Benítez-Del-Castillo, J M

2018-03-01

Non-interventional, observational, epidemiology study to assess clinical characteristics and frequency of adenovirus conjunctivitis diagnosed by AdenoPlus ® in patients who presented with signs and symptoms of acute conjunctivitis. Safety aspects during use of the test were analyzed as well. This analysis presents the data obtained from the 386 patients enrolled in Spain. Patients had to present with acute signs and symptoms of conjunctivitis ≤7days. The minimum age was 1year old. Patients who had already used local antiviral therapies, topical steroids or immuno-modulators were not allowed to enter the study. A standardized questionnaire was used to collect patient's ocular history and test results. A total of 386 patients were recruited in 22 sites, being analyzed 329 patients. Among them, the percentage of "AdenoPlus ® positive" was 36,2% (119/329). Before the test was performed, in 84,1% of the cases, investigators believed that the conjunctivitis was of viral origin but only 50,3% of the investigators had their clinical assessment confirmed by the test. Patients who tested positive for adenoviral conjunctivitis presented higher percentages of signs and symptoms than the rest of the patients. None of the signs or symptoms could be qualified as pathognomonic of the disease, being difficult for the clinicians to perform an accurate diagnosis. AdenoPlus ® test is an antigen based immunoassay test that detects the presence of adenovirus directly from tears, and it can be an useful tool to help early differential diagnosis in patients with conjunctivitis signs and symptoms lasting for less than or equal to 7days. Copyright © 2017 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

Membrane dynamics associated with viral infection.

PubMed

de Armas-Rillo, Laura; Valera, María-Soledad; Marrero-Hernández, Sara; Valenzuela-Fernández, Agustín

2016-05-01

Viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. These processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the ER, mitochondria, Golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. In fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. In this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. By defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © 2015 The Authors Reviews in Medical Virology Published by John Wiley & Sons, Ltd.

Molecular analysis of the genes involved in the biosynthesis of serotype specific polysaccharide in the novel serotype k strains of Streptococcus mutans.

PubMed

Nomura, R; Nakano, K; Ooshima, T

2005-10-01

We previously reported the new serotype k of Streptococcus mutans, which, compared to serotypes c, e, and f, features a drastic reduction in the length of the glucose side chain linked to the rhamnose backbone of the serotype specific polysaccharide. The 5' region of the rgpF gene of serotype k strains contains a distinctive nucleotide sequence, which suggests that an alteration of the rgpF gene in serotype k strains may explain the shortened glucose side chain. However, in the present study, expression of the rgpF gene of MT8148 (serotype c) in serotype k isolates was not found to lead to serotype conversion. Furthermore, mRNA expression of rgpE, known to be associated with glucose side chain formation, was not detected in any of the tested serotype k isolates with an RT-PCR method. The nucleotide alignment of all genes known to be involved in the biosynthesis of serotype specific polysaccharide in serotype k strains was shown to be quite similar to that of serotype c strains, as compared to serotype e and f strains, especially in the region downstream of rgpF. Our results indicate that the common characteristics of serotype k isolates may be caused by a lack of expression of the gene involved in glucose side chain formation.

Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.

PubMed

Matsuzaki, Yasunori; Konno, Ayumu; Mochizuki, Ryuta; Shinohara, Yoichiro; Nitta, Keisuke; Okada, Yukihiro; Hirai, Hirokazu

2018-02-05

Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken β-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0-2%) and astrocytes (0.1-2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative biology of rAAV transduction in ferret, pig and human airway epithelia.

PubMed

Liu, X; Luo, M; Guo, C; Yan, Z; Wang, Y; Engelhardt, J F

2007-11-01

Differences between rodent and human airway cell biology have made it difficult to translate recombinant adeno-associated virus (rAAV)-mediated gene therapies to the lung for cystic fibrosis (CF). As new ferret and pig models for CF become available, knowledge about host cell/vector interactions in these species will become increasingly important for testing potential gene therapies. To this end, we have compared the transduction biology of three rAAV serotypes (AAV1, 2 and 5) in human, ferret, pig and mouse-polarized airway epithelia. Our results indicate that apical transduction of ferret and pig airway epithelia with these rAAV serotypes closely mirrors that observed in human epithelia (rAAV1>rAAV2 congruent withrAAV5), while transduction of mouse epithelia was significantly different (rAAV1>rAAV5>rAAV2). Similarly, ferret, pig and human epithelia also shared serotype-specific differences in the polarity (apical vs basolateral) and proteasome dependence of rAAV transduction. Despite these parallels, N-linked sialic acid receptors were required for rAAV1 and rAAV5 transduction of human and mouse airway epithelia, but not ferret or pig airway epithelia. Hence, although the airway tropisms of rAAV serotypes 1, 2 and 5 are conserved better among ferret, pig and human as compared to mouse, viral receptors/co-receptors appear to maintain considerable species diversity.

A review of therapeutic prospects of non-viral gene therapy in the retinal pigment epithelium

PubMed Central

Koirala, Adarsha; Conley, Shannon M.; Naash, Muna I.

2013-01-01

Ocular gene therapy has been extensively explored in recent years as a therapeutic avenue to target diseases of the cornea, retina and retinal pigment epithelium (RPE). Adeno-associated virus (AAV)-mediated gene therapy has shown promise in several RPE clinical trials but AAVs have limited payload capacity and potential immunogenicity. Traditionally however, non-viral alternatives have been plagued by low transfection efficiency, short-term expression and low expression levels. Recently, these drawbacks have begun to be overcome by the use of specialty carriers such as polylysine, liposomes, or polyethyleneimines, and by inclusion of suitable DNA elements to enhance gene expression and longevity. Recent advancements in the field have yielded non-viral vectors that have favorable safety profiles, lack immunogenicity, exhibit long-term elevated gene expression, and show efficient transfection in the retina and RPE, making them poised to transition to clinical applications. Here we discuss the advancements in nanotechnology and vector engineering that have improved the prospects for clinical application of non-viral gene therapy in the RPE. PMID:23796578

Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.

PubMed

Roche-Molina, Marta; Sanz-Rosa, David; Cruz, Francisco M; García-Prieto, Jaime; López, Sergio; Abia, Rocío; Muriana, Francisco J G; Fuster, Valentín; Ibáñez, Borja; Bernal, Juan A

2015-01-01

Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels. Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses. © 2014 American Heart Association, Inc.

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

PubMed

Brockstedt, D G; Podsakoff, G M; Fong, L; Kurtzman, G; Mueller-Ruchholtz, W; Engleman, E G

1999-07-01

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations. Copyright 1999 Academic Press.

Cytolethal distending toxin in isolates of Aggregatibacter actinomycetemcomitans from Ghanaian adolescents and association with serotype and disease progression.

PubMed

Höglund Åberg, Carola; Antonoglou, Georgios; Haubek, Dorte; Kwamin, Francis; Claesson, Rolf; Johansson, Anders

2013-01-01

The cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL). A total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up. Presence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994-8.779], p<0.001). A. actinomycetemcomitans isolated from the Ghanaian adolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.

Characterization of Chinese Haemophilus parasuis Isolates by Traditional Serotyping and Molecular Serotyping Methods

PubMed Central

Ma, Lina; Wang, Liyan; Chu, Yuefeng; Li, Xuerui; Cui, Yujun; Chen, Shengli; Zhou, Jianhua; Li, Chunling; Lu, Zhongxin; Liu, Jixing; Liu, Yongsheng

2016-01-01

Haemophilus parasuis is classified mainly through serotyping, but traditional serotyping always yields non-typable (NT) strains and unreliable results via cross-reactions. Here, we surveyed the serotype prevalence of Chinese H. parasuis isolates using traditional serotyping (gel immuno-diffusion test, GID) and molecular serotyping (multiplex PCR, mPCR). We also investigated why discrepant results between these methods were obtained, and investigated mPCR failure through whole-genome sequencing. Of the 100 isolate tested, 73 (73%) and 93 (93%) were serotyped by the GID test and mPCR, respectively, with a concordance rate of 66% (66/100). Additionally, mPCR reduced the number of NT isolates from 27 (27%) for the GID testing, to seven (7%). Eleven isolates were sequenced, including nine serotype-discrepant isolates from mPCR and GID typing (excluding strains that were NT by GID only) and two NT isolates from both methods, and their in silico serotypes were obtained from genome sequencing based on their capsule loci. The mPCR results were supported by the in silico serotyping of the seven serotype-discrepant isolates. The discrepant results and NT isolates determined by mPCR were attributed to deletions and unknown sequences in the serotype-specific region of each capsule locus. Compared with previous investigations, this study found a similar predominant serotype profile, but a different prevalence frequency for H. parasuis, and the five most prevalent serotypes or strain groups were serotypes 5, 4, NT, 7 and 13 for mPCR, and serotypes 5, NT, 4, 7 and 13/10/14 for GID. Additionally, serotype 7 was recognized as a principal serotype in this work. PMID:28005999

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus.

PubMed

Watson, G L; Sayles, J N; Chen, C; Elliger, S S; Elliger, C A; Raju, N R; Kurtzman, G J; Podsakoff, G M

1998-12-01

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a genetic deficiency of beta-glucuronidase (GUS). We used a recombinant adeno-associated virus vector (AAV-GUS) to deliver GUS cDNA to MPS VII mice. The route of vector administration had a dramatic effect on the extent and distribution of GUS activity. Intramuscular injection of AAV-GUS resulted in high, localized production of GUS, while intravenous administration produced low GUS activity in several tissues. This latter treatment of MPS VII mice reduced glycosaminoglycan levels in the liver to normal and reduced storage granules dramatically. We show that a single administration of AAV-GUS can provide sustained expression of GUS in a variety of cell types and is sufficient to reverse the disease phenotype at least in the liver.

Postentry processing of recombinant adeno-associated virus type 1 and transduction of the ferret lung are altered by a factor in airway secretions.

PubMed

Yan, Ziying; Sun, Xingshen; Evans, Idil A; Tyler, Scott R; Song, Yi; Liu, Xiaoming; Sui, Hongshu; Engelhardt, John F

2013-09-01

We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (~1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion.

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

PubMed

Valdor, Markus; Wagner, Anke; Röhrs, Viola; Berg, Johanna; Fechner, Henry; Schröder, Wolfgang; Tzschentke, Thomas M; Bahrenberg, Gregor; Christoph, Thomas; Kurreck, Jens

2018-01-01

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal

PubMed Central

Gupta, Birendra Prasad; Singh, Sneha; Kurmi, Roshan; Malla, Rajani; Sreekumar, Easwaran; Manandhar, Krishna Das

2015-01-01

Background & objectives: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. Methods: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. Results: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. Interpretations & conclusions: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region. PMID:26905233

An experimental and computational evolution-based method to study a mode of co-evolution of overlapping open reading frames in the AAV2 viral genome.

PubMed

Kawano, Yasuhiro; Neeley, Shane; Adachi, Kei; Nakai, Hiroyuki

2013-01-01

Overlapping open reading frames (ORFs) in viral genomes undergo co-evolution; however, how individual amino acids coded by overlapping ORFs are structurally, functionally, and co-evolutionarily constrained remains difficult to address by conventional homologous sequence alignment approaches. We report here a new experimental and computational evolution-based methodology to address this question and report its preliminary application to elucidating a mode of co-evolution of the frame-shifted overlapping ORFs in the adeno-associated virus (AAV) serotype 2 viral genome. These ORFs encode both capsid VP protein and non-structural assembly-activating protein (AAP). To show proof of principle of the new method, we focused on the evolutionarily conserved QVKEVTQ and KSKRSRR motifs, a pair of overlapping heptapeptides in VP and AAP, respectively. In the new method, we first identified a large number of capsid-forming VP3 mutants and functionally competent AAP mutants of these motifs from mutant libraries by experimental directed evolution under no co-evolutionary constraints. We used Illumina sequencing to obtain a large dataset and then statistically assessed the viability of VP and AAP heptapeptide mutants. The obtained heptapeptide information was then integrated into an evolutionary algorithm, with which VP and AAP were co-evolved from random or native nucleotide sequences in silico. As a result, we demonstrate that these two heptapeptide motifs could exhibit high degeneracy if coded by separate nucleotide sequences, and elucidate how overlap-evoked co-evolutionary constraints play a role in making the VP and AAP heptapeptide sequences into the present shape. Specifically, we demonstrate that two valine (V) residues and β-strand propensity in QVKEVTQ are structurally important, the strongly negative and hydrophilic nature of KSKRSRR is functionally important, and overlap-evoked co-evolution imposes strong constraints on serine (S) residues in KSKRSRR, despite high

Viral-bacterial associations in acute apical abscesses.

PubMed

Ferreira, Dennis C; Rôças, Isabela N; Paiva, Simone S M; Carmo, Flávia L; Cavalcante, Fernanda S; Rosado, Alexandre S; Santos, Kátia R N; Siqueira, José F

2011-08-01

Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses. Copyright © 2011 Mosby, Inc. All rights reserved.

Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System

PubMed Central

Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe

2015-01-01

Abstract Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

Ureaplasma serotypes associated with the bovine urogenital tract.

PubMed Central

Truscott, R B

1983-01-01

One hundred and thirty-one ureaplasma isolates were tested using the immunoperoxidase system. Thirty-four were from semen, 34 from preputial washes of normal bulls and 63 were from vaginal swabs from herds experiencing infertility problems and/or vulvovaginitis. The serotypes from semen were T44 (12.1%), Bu2 (11.2%), D48 (2.8%), T315 (0.9%) and T288 (0.9%). Those from preputial washes were T44 (9.3%), Bu2 (8.4%), T288 (7.5%), D48 (0.9%) and T95 (0.9%). From vaginal swabs the serotypes were D48 (22.4%), Bu2 (10.3%), T45 (4.7%), T288 (3.8%) and T315 (1.9%). PMID:6365295

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

PubMed Central

Li, Chengwen; He, Yi; Nicolson, Sarah; Hirsch, Matt; Weinberg, Marc S.; Zhang, Ping; Kafri, Tal; Samulski, R. Jude

2013-01-01

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials. PMID:23454772

Quinolone-resistant Salmonella enterica serotype Enteritidis infections associated with international travel.

PubMed

O'Donnell, Allison T; Vieira, Antonio R; Huang, Jennifer Y; Whichard, Jean; Cole, Dana; Karp, Beth E

2014-11-01

We found a strong association between nalidixic acid-resistant Salmonella enterica serotype Enteritidis infections in the United States and recent international travel by linking Salmonella Enteritidis data from the National Antimicrobial Resistance Monitoring System and the Foodborne Diseases Active Surveillance Network. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

[Dengue virus serotype 1 (DENV-1) from Colombia: its contribution to dengue occurrence in Santander].

PubMed

Ocazionez-Jiménez, Raquel E; Ortiz-Báez, Ayda Susana; Gómez-Rangel, Sergio Yebrail; Miranda-Esquivel, Daniel R

2013-09-01

Between 1998 and 2008 all dengue virus serotypes circulated in the Departamento de Santander, an endemic region in northeastern Colombia. No information is available as to the role of serotype 1 (DENV-1) with respect to epidemiology of dengue. To analyze the relationship between changes in DENV-1 predominance with respect to genetic diversity, prevalence of others serotypes and occurrence of severe dengue. Virus genetic diversity was studied by phylogenetic analysis comparing E gene sequences from 12 viral strains. Data about serotypes predominance obtained in previous studies and official data about dengue incidence were used for analysis. Selected viruses grouped into genotype V together DENV-1 from Latin America countries, and segregation in four lineages was evidenced. Changes in virus predominance coincided with replacement of lineage, increase in prevalence of DENV-2 and DENV-3 and increase of severe dengue. Genetic divergence could have contributed to changes in DENV-1 predominance. The relationship of the virus with DENV-2 and DENV-3 could create scenarios that promote occurrence of severe cases. More studies are required to ascertain the precise role of serotypes in the epidemiology of dengue.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

PubMed

Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

2009-08-01

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

Human beta defensin-1 is involved in the susceptibility to adeno-tonsillar hypertrophy.

PubMed

Zupin, Luisa; Celsi, Fulvio; Bresciani, Martina; Orzan, Eva; Grasso, Domenico Leonardo; Crovella, Sergio

2018-04-01

Innate immunity molecules are known to play a pivotal role in the homeostasis of the oral mucosa, permitting the presence of commensal microflora and, at the same time, providing a first line of defense against pathogens attempting to invade the oral cavity. Tonsils represent the local immune tissue in oral cavity, being able to provide a non-specific response to pathogens; however, in the presence of microbes or foreign materials present in the mouth tonsils could became infected and develop chronic inflammation, thus leading to hypertrophy. The etiology of the disease is multifactorial depending upon environmental and host factors, the latter including molecules of mucosal innate immunity. Ninety-five children with adeno-tonsillar hypertrophy subjected to adeno-tonsillectomy were recruited at the pediatric otorhinolaryngology service of the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste (Italy). The specimen discarded from the surgery were used for genomic DNA extraction and genotyping, for mRNA extraction and gene expression analysis, finally the samples were cut and used to prepare slides to perform immunohistochemistry. Functional polymorphisms within DEFB1 gene, encoding the human beta defensin-1 (hBD-1), were analyzed finding association between DEFB1 rare haplotypes and susceptibility to adeno-tonsillar hypertrophy. DEFB1 mRNA expression was detected in the tonsils and the hBD-1 protein was localized at the epithelia of tonsils mainly in the proximity of the basal lamina. Our findings lead us to hypothesize an involvement of hBD-1 mediated innate immunity in the modulation of the susceptibility towards adeno-tonsillar hypertrophy development. Copyright © 2018 Elsevier B.V. All rights reserved.

Nucleotide substitutions in dengue virus serotypes from Asian and American countries: insights into intracodon recombination and purifying selection

PubMed Central

2013-01-01

Background Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood. Results We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus. Conclusion The study provides the first evidence for intracodon

Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

PubMed Central

Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

2008-01-01

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, Tonie E.; Smith, Susan R.; Miyamoto, Amy; Shadduck, Daniel J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

USGS Publications Warehouse

Rocke, T.E.; Smith, S.R.; Miyamoto, A.; Shadduck, D.J.

2002-01-01

A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

Viral Vectors for Gene Delivery to the Central Nervous System

PubMed Central

Lentz, Thomas B.; Gray, Steven J.; Samulski, R. Jude

2011-01-01

The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features. PMID:22001604

[Dengue hemorrhagic fever serotype and infection pattern in a Colombian endemic area].

PubMed

Ocazionez, Raquel E; Gómez, Sergio Y; Cortés, Fabián M

2007-01-01

Describing the relationship between viral serotypes, infection pattern and dengue hemorrhagic fever. 1,545 febrile patients were studied from 1998-2004 in the Santander department of Colombia. Dengue infection was confirmed by IgM ELISA and the virus was isolated in C6/36 cells. Infection pattern was established by detecting IgG antibodies in acute serum. Neutralising antibody titres were investigated in dengue cases occurring during years when less (1998) and more (2001) dengue hemorrhagic cases were reported by using PRNT. DEN-1 predominance in 1998 and the re-introduction of DEN-3 in 2001 coincided with an epidemic. DEN-2 infection caused more hemorrhagic cases than DEN-3 infection (24,5 % cf 11,2 %; p<0.05). DEN-2 was more associated with secondary infection than DEN-3 (56,8 % cf 15,7 %; p<0.001). An annual decrease of DHF was correlated with decreased DEN-2 dominance (r=0.95; p= 0.01), and secondary infection (r=0.9; p=0.03) and increased DEN-3 predominance (r=-0.91; p=0.03). There were no differences in neutralising antibody titres amongst analysed cases. DEN-1 neutralising antibodies presented the highest titres. Change in relative dengue virus serotype abundance was associated with changed infection pattern and DHF frequency. Continuing virological surveillance should become a priority for preventing dengue hemorrhagic fever in endemic areas.

Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandramouli, Sumana; Joseph, Jeremiah S.; Daudenarde, Sophie

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication bymore » processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-{angstrom} crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.« less

Imported dengue virus serotype 1 from Madeira to Finland 2012.

PubMed

Huhtamo, E; Korhonen, Em; Vapalahti, O

2013-02-21

Imported dengue cases originating from the Madeiran outbreak are increasingly reported. In 2012 five Finnish travellers returning from Madeira were diagnosed with dengue fever. Viral sequence data was obtained from two patients. The partial C-preM sequences (399 and 396 bp respectively) were found similar to that of an autochthonous case from Madeira. The partial E-gene sequence (933 bp) which was identical among the two patients grouped phylogenetically with South American strains of dengue virus serotype 1.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

Adeno-associated virus vectors can be efficiently produced without helper virus.

PubMed

Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

1998-07-01

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

PubMed Central

Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

2013-01-01

Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

Vector competence of the Aedes aegypti population from Santiago Island, Cape Verde, to different serotypes of dengue virus.

PubMed

da Moura, Aires Januário Fernandes; de Melo Santos, Maria Alice Varjal; Oliveira, Claudia Maria Fontes; Guedes, Duschinka Ribeiro Duarte; de Carvalho-Leandro, Danilo; da Cruz Brito, Maria Lidia; Rocha, Hélio Daniel Ribeiro; Gómez, Lara Ferrero; Ayres, Constância Flávia Junqueira

2015-02-19

Dengue is an arboviral disease caused by dengue virus (DENV), whose main vectors are the mosquitoes Aedes aegypti and Aedes albopictus. A. aegypti is the only DENV vector in Cape Verde, an African country that suffered its first outbreak of dengue in 2009. However, little is known about the variation in the level of vector competence of this mosquito population to the different DENV serotypes. This study aimed to evaluate the vector competence of A. aegypti from the island of Santiago, Cape Verde, to four DENV serotypes and to detect DENV vertical transmission. Mosquitoes were fed on blood containing DENV serotypes and were dissected at 7, 14 and 21 days post-infection (dpi) to detect the virus in the midgut, head and salivary glands (SG) using RT-PCR. Additionally, the number of copies of viral RNA present in the SG was determined by qRT-PCR. Furthermore, eggs were collected in the field and adult mosquitoes obtained were analyzed by RT-PCR and the platelia dengue NS1 antigen kit to detect transovarial transmission. High rates of SG infection were observed for DENV-2 and DENV-3 whereas for DENV-1, viral RNA was only detected in the midgut and head. DENV-4 did not spread to the head or SG, maintaining the infection only in the midgut. The number of viral RNA copies in the SG did not vary significantly between DENV-2 and DENV-3 or among the different periods of incubation and the various titers of DENV tested. With respect to DENV surveillance in mosquitoes obtained from the eggs collected in the field, no samples were positive. Although no DENV positive samples were collected from the field in 2014, it is important to highlight that the A. aegypti population from Santiago Islands exhibited different degrees of susceptibility to DENV serotypes. This population showed a high vector competence for DENV-2 and DENV-3 strains and a low susceptibility to DENV-1 and DENV-4. Viral RNA copies in the SG remained constant for at least 21 dpi, which may enhance the vector

Natural Infections With Pigeon Paramyxovirus Serotype 1: Pathologic Changes in Eurasian Collared-Doves ( Streptopelia decaocto) and Rock Pigeons ( Columba livia) in the United States.

PubMed

Isidoro-Ayza, M; Afonso, C L; Stanton, J B; Knowles, S; Ip, H S; White, C L; Fenton, H; Ruder, M G; Dolinski, A C; Lankton, J

2017-07-01

Pigeon paramyxovirus serotype 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus serotype 1 serogroup that causes mortality in columbiformes and poultry. Following introduction into the United States in the mid-1980s, PPMV-1 rapidly spread causing numerous mortality events in Eurasian collared-doves ( Streptopelia decaocto) (ECDOs) and rock pigeons ( Columba livia) (ROPIs). The investigators reviewed pathological findings of 70 naturally infected, free-ranging columbiforms from 25 different mortality events in the United States. Immunohistochemistry targeting PPMV-1 nucleoprotein was used to determine the tissue distribution of the virus in a subset of 17 birds from 10 of the studied outbreaks. ECDOs (61 birds) and ROPIs (9 birds) were the only species in which PPMV-1-associated disease was confirmed by viral isolation and presence of histologic lesions. Acute to subacute tubulointerstitial nephritis and necrotizing pancreatitis were the most frequent histologic lesions, with immunolabeling of viral antigen in renal tubular epithelial cells and pancreatic acinar epithelium. Lymphoid depletion of bursa of Fabricius and spleen was common, but the presence of viral antigen in these organs was inconsistent among infected birds. Hepatocellular necrosis was occasionally present with immunolabeling of hypertrophic Kupffer cells, and immunopositive eosinophilic intracytoplasmic inclusion bodies were present in hepatocytes of 1 ECDO. Immunopositive lymphocytic choroiditis was present in 1 ECDO, while lymphocytic meningoencephalitis was frequent in ROPIs in absence of immunolabeling. This study demonstrates widespread presence of PPMV-1 antigen in association with histologic lesions, confirming the lethal potential of this virus in these particular bird species.

Natural infections with pigeon paramyxovirus serotype 1: Pathologic changes in Eurasian collared-doves (Streptopelia decaocto) and rock pigeons (Columba livia) in the United States

USGS Publications Warehouse

Isidoro Ayza, Marcos; Afonso, C.L.; Stanton, J.B.; Knowles, Susan N.; Ip, Hon S.; White, C. LeAnn; Fenton, Heather; Ruder, M.G.; Dolinski, A. C.; Lankton, Julia S.

2017-01-01

Pigeon paramyxovirus serotype 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus serotype 1 serogroup that causes mortality in columbiformes and poultry. Following introduction into the United States in the mid-1980s, PPMV-1 rapidly spread causing numerous mortality events in Eurasian collared-doves (Streptopelia decaocto) (ECDOs) and rock pigeons (Columba livia) (ROPIs). The investigators reviewed pathological findings of 70 naturally infected, free-ranging columbiforms from 25 different mortality events in the United States. Immunohistochemistry targeting PPMV-1 nucleoprotein was used to determine the tissue distribution of the virus in a subset of 17 birds from 10 of the studied outbreaks. ECDOs (61 birds) and ROPIs (9 birds) were the only species in which PPMV-1-associated disease was confirmed by viral isolation and presence of histologic lesions. Acute to subacute tubulointerstitial nephritis and necrotizing pancreatitis were the most frequent histologic lesions, with immunolabeling of viral antigen in renal tubular epithelial cells and pancreatic acinar epithelium. Lymphoid depletion of bursa of Fabricius and spleen was common, but the presence of viral antigen in these organs was inconsistent among infected birds. Hepatocellular necrosis was occasionally present with immunolabeling of hypertrophic Kupffer cells, and immunopositive eosinophilic intracytoplasmic inclusion bodies were present in hepatocytes of 1 ECDO. Immunopositive lymphocytic choroiditis was present in 1 ECDO, while lymphocytic meningoencephalitis was frequent in ROPIs in absence of immunolabeling. This study demonstrates widespread presence of PPMV-1 antigen in association with histologic lesions, confirming the lethal potential of this virus in these particular bird species.

In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

PubMed Central

Lau, Cia-Hin; Suh, Yousin

2017-01-01

Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics. PMID:29333255

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution.

PubMed

Ippolito, Danielle L; James, Wesley A; Tinnemore, Deborah; Huang, Raywin R; Dehart, Mary J; Williams, Julie; Wingerd, Mark A; Demons, Samandra T

2010-11-24

Group B Streptococcus (GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence. Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005). This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution

PubMed Central

2010-01-01

Background Group B Streptococcus (GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. Methods To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence. Results Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005). Conclusion This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades. PMID:21106080

Full genome sequence of the first bluetongue virus serotype 21 (BTV-21) isolated from China: evidence for genetic reassortment between BTV-21 and bluetongue virus serotype 16 (BTV-16).

PubMed

Qin, Shaomin; Yang, Heng; Zhang, Yixuan; Li, Zhanhong; Lin, Jun; Gao, Lin; Liao, Defang; Cao, Yingying; Ren, Pengfei; Li, Huachun; Wu, Jianmin

2018-05-01

Bluetongue (BT) is one of the most important insect-borne, non-contagious viral diseases of ruminants and can cause severe disease and death in sheep. Its pathogen, bluetongue virus (BTV) has a double-stranded RNA genome consisting of 10 segments that provides an opportunity for field and vaccine strains of different serotypes to reassort whilst simultaneously infecting the same animal. For the first time, we report the full-length genome sequence of a BTV strain of serotype 21 (5149E) isolated from sentinel cattle in Guangxi Province in China in 2015. Sequence analysis suggested that the isolate 5149E had undergone a reassortment incident and acquired seg-6 from an isolate of BTV-16 which originated from Japan. This study aims to provide more understanding as to the origin and epidemiology of BTV.

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

PubMed

Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

The B Cell Response to Foot-and-Mouth Disease Virus in Cattle following Sequential Vaccination with Multiple Serotypes.

PubMed

Grant, Clare F J; Carr, B Veronica; Kotecha, Abhay; van den Born, Erwin; Stuart, David I; Hammond, John A; Charleston, Bryan

2017-05-01

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid. IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV. Copyright © 2017 American Society for Microbiology.

Salmonella serotypes in reptiles and humans, French Guiana.

PubMed

Gay, Noellie; Le Hello, Simon; Weill, François-Xavier; de Thoisy, Benoit; Berger, Franck

2014-05-14

In French Guiana, a French overseas territory located in the South American northern coast, nearly 50% of Salmonella serotypes isolated from human infections belong to serotypes rarely encountered in metropolitan France. A reptilian source of contamination has been investigated. Between April and June 2011, in the area around Cayenne, 151 reptiles were collected: 38 lizards, 37 snakes, 32 turtles, 23 green iguanas and 21 caimans. Cloacal swab samples were collected and cultured. Isolated Salmonella strains were identified biochemically and serotyped. The overall carriage frequency of carriage was 23.2% (95% confidence interval: 16.7-30.4) with 23 serotyped strains. The frequency of Salmonella carriage was significantly higher for wild reptiles. Near two-thirds of the Salmonella serotypes isolated from reptiles were also isolated from patients in French Guiana. Our results highlight the risk associated with the handling and consumption of reptiles and their role in the spread of Salmonella in the environment. Copyright © 2014 Elsevier B.V. All rights reserved.

An "on-matrix" digestion procedure for AP-MS experiments dissects the interplay between complex-conserved and serotype-specific reactivities in Dengue virus-human plasma interactome.

PubMed

Ramos, Yassel; Huerta, Vivian; Martín, Dayron; Palomares, Sucel; Yero, Alexis; Pupo, Dianne; Gallien, Sebastien; Martín, Alejandro M; Pérez-Riverol, Yasset; Sarría, Mónica; Guirola, Osmany; Chinea, Glay; Domon, Bruno; González, Luis Javier

2017-07-13

The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here. This procedure enabled the identification of 16 human plasma proteins interacting with domain III from the envelope protein of DENV serotypes 1, 3 and 4 that would have not been detected otherwise and increased the known DIIIE interactors in human plasma to 59 proteins. Selected Reaction Monitoring analysis evidenced DENV interactome in human plasma is rather conserved although significant differences on the reactivity of viral serotypes with specific proteins do exist. A comparison between the serotype-dependent profile of reactivity and the conservation pattern of amino acid residues suggests an evolutionary selection of highly conserved interactions with the host and other interactions mediated for surface regions of higher variability. False negative results on the identification of interacting proteins in pull-down experiments compromise the subsequent interpretation of results and the formulation of a working hypothesis for the derived future work. In this study we demonstrate the presence of bait-interacting proteins reluctant to dissociate under elution conditions of acid pH and presence of chaotropics. We propose the direct proteolytic digestion of proteins while still bound to the affinity matrix ("on-matrix" digestion) and evaluate the impact of this methodology in the comparative study of the interactome of the four serotypes of Dengue virus mediated by

Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China.

PubMed

Zhang, Teng; Jin, Qianyue; Ding, Peiyang; Wang, Yinbiao; Chai, Yongxiao; Li, Yafei; Liu, Xiao; Luo, Jun; Zhang, Gaiping

2016-11-18

In several parts of China, there have been a large number of hydropericardium syndrome (HPS) outbreaks caused by serotype 4 fowl adenovirus (FAdV-4) in broiler chickens since 2015. These outbreak-associated FAdV-4 strains were distinct from previous circulating strains which did not lead to severe HPS outbreaks. To better understand the molecular epidemiology of the currently circulating FAdV strains for effective diagnosis and treatment of HPS, we isolated 12 HPS outbreak-associated FAdV-4 strains from different regions in central China and investigated their molecular characteristics by performing phylogenetic analyses based on the hexon genes. Our results indicated the FAdV-4 strains in this study all belonged to serotype FAdV-4, species FAdV-C. And in comparison with ON1, KR5, MX-SHP95, PK-01, PJ-06 strains within the cluster where outbreak-associated FAdV-4 strains were located, the nucleotide sequence divergence were 1.31, 1.10, 1.42, 2.77 and 2.84%, respectively. Phylogenetic analyses revealed the hexon genes of the 12 outbreak-associated strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor and we suggested that these outbreak-associated FAdV-4 strains originate from earlier strains in India.

Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

PubMed

Hastie, Eric; Samulski, R Jude

2015-05-01

Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

Evaluation of cross-protection of bluetongue virus serotype 4 with other serotypes in sheep.

PubMed

Zulu, Gcwalisile B; Venter, Estelle H

2014-10-16

Bluetongue (BT) is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV). Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control), 1 (unrelated serotype), 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes.

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

PubMed

Messer, William B; Yount, Boyd L; Royal, Scott R; de Alwis, Ruklanthi; Widman, Douglas G; Smith, Scott A; Crowe, James E; Pfaff, Jennifer M; Kahle, Kristen M; Doranz, Benjamin J; Ibarra, Kristie D; Harris, Eva; de Silva, Aravinda M; Baric, Ralph S

2016-05-15

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.

PubMed

Vallery, Tenaya K; Withers, Johanna B; Andoh, Joana A; Steitz, Joan A

2018-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments. IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus. Copyright © 2018 American Society for Microbiology.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

PubMed

Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption

PubMed Central

Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae

2013-01-01

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications, but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions. PMID:23899192

AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.

PubMed

Huang, Xinping; Hartley, Antja-Voy; Yin, Yishi; Herskowitz, Jeremy H; Lah, James J; Ressler, Kerry J

2013-11-01

The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 μg DNA per ml of media (21 μg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer. Copyright © 2013 Elsevier B.V. All rights reserved.

A Novel Avian Paramyxovirus (Putative Serotype 15) Isolated from Wild Birds

PubMed Central

Lee, Hyun-Jeong; Kim, Ji-Ye; Lee, Youn-Jeong; Lee, Eun-Kyung; Song, Byoung-Min; Lee, Hee-Soo; Choi, Kang-Seuk

2017-01-01

In January 2014, a viral hemagglutinating agent named UPO216 was isolated from fecal droppings of wild birds at the UPO wetland in South Korea during an avian influenza surveillance program. Electron microscopy identified the UPO216 virus as an avian paramyxovirus (APMV). Pathogenicity tests and molecular pathotyping revealed that the virus was avirulent in chickens. The UPO216 virus was assigned to a serological group antigenically distinct from known serotypes of APMV (−1, −2, −3, −4, −6, −7, −8, and −9) by hemagglutination inhibition test, despite showing weak cross-reactivity with APMV-1 and APMV-9. The UPO216 virus RNA genome is 15,180 nucleotides (nts) in length, encodes 3′-N-P(V/W)-M-F-HN-L-5′ in that order, and shows unique genetic characteristics in terms of genomic composition and evolutionary divergence (0.43 or greater from known serotypes of APMV). Phylogenetic analysis revealed that the UPO216 occupies a branch separate from APMV-1, -9, -12, and -13. Serologic surveillance of wild birds (n = 880; 15 species, five Orders) detected UPO216-reactive antibodies in 4% (20/494) of serum samples taken from five species of wild duck belonging to the Order Anseriformes. In particular, UPO216-specific antibodies showing no cross-reaction with other serotypes of APMV were detected in four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). These results indicate that the UPO216 virus has antigenically and genetically unique characteristics distinct from known serotypes of APMV and likely has been circulating widely in wild duck species of the Order Anseriformes. Thus, we propose the UPO216 isolate as a prototype strain of a novel APMV serotype (putative APMV-15). PMID:28529504

A Novel Avian Paramyxovirus (Putative Serotype 15) Isolated from Wild Birds.

PubMed

Lee, Hyun-Jeong; Kim, Ji-Ye; Lee, Youn-Jeong; Lee, Eun-Kyung; Song, Byoung-Min; Lee, Hee-Soo; Choi, Kang-Seuk

2017-01-01

In January 2014, a viral hemagglutinating agent named UPO216 was isolated from fecal droppings of wild birds at the UPO wetland in South Korea during an avian influenza surveillance program. Electron microscopy identified the UPO216 virus as an avian paramyxovirus (APMV). Pathogenicity tests and molecular pathotyping revealed that the virus was avirulent in chickens. The UPO216 virus was assigned to a serological group antigenically distinct from known serotypes of APMV (-1, -2, -3, -4, -6, -7, -8, and -9) by hemagglutination inhibition test, despite showing weak cross-reactivity with APMV-1 and APMV-9. The UPO216 virus RNA genome is 15,180 nucleotides (nts) in length, encodes 3'-N-P(V/W)-M-F-HN-L-5' in that order, and shows unique genetic characteristics in terms of genomic composition and evolutionary divergence (0.43 or greater from known serotypes of APMV). Phylogenetic analysis revealed that the UPO216 occupies a branch separate from APMV-1, -9, -12, and -13. Serologic surveillance of wild birds ( n = 880; 15 species, five Orders) detected UPO216-reactive antibodies in 4% (20/494) of serum samples taken from five species of wild duck belonging to the Order Anseriformes . In particular, UPO216-specific antibodies showing no cross-reaction with other serotypes of APMV were detected in four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). These results indicate that the UPO216 virus has antigenically and genetically unique characteristics distinct from known serotypes of APMV and likely has been circulating widely in wild duck species of the Order Anseriformes . Thus, we propose the UPO216 isolate as a prototype strain of a novel APMV serotype (putative APMV-15).

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

PubMed Central

Scharinger, Eva J.; Dietrich, Richard; Kleinsteuber, Ina; Märtlbauer, Erwin

2016-01-01

Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind to C. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 103 and 9 × 106 CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacteriaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method. PMID:26850303

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil.

PubMed

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Watanabe, Aripuanã Sakurada Aranha; Biselli-Périco, Joice Matos; Drumond, Betânia Paiva; Ribeiro, Milene Rocha; Vedovello, Danila; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; Chaves, Bárbara Aparecida; Silva, Mayara Marques Carneiro da; Batista, Izabella Cristina Andrade; Papa, Michelle Premazzi; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; Arruda, Luciana Barros de; Souza, Danielle da Glória de; Teixeira, Mauro Martins; Vasilakis, Nikos; Nogueira, Maurício Lacerda

2018-05-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto.

Viral immunogenicity determines epidemiological fitness in a cohort of DENV-1 infection in Brazil

PubMed Central

Pinheiro, Tauyne Menegaldo; Mota, Mânlio Tasso de Oliveira; Biselli-Périco, Joice Matos; Ribeiro, Milene Rocha; Araújo, João Pessoa; Pimenta, Paulo Filemon Paolucci; da Silva, Mayara Marques Carneiro; Batista, Izabella Cristina Andrade; Meuren, Lana Monteiro; Lucas, Carolina Gonçalves de Oliveira; Matassoli, Flavio Lemos; Gil, Laura Helena Vega Gonzales; Bozzi, Adriana; Calzavara-Silva, Carlos Eduardo; de Souza, Danielle da Glória; Teixeira, Mauro Martins; Vasilakis, Nikos

2018-01-01

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto. PMID:29813061

Induction of Broad-Spectrum Protective Immunity against Disparate Cryptococcus Serotypes

PubMed Central

Van Dyke, Marley C. Caballero; Chaturvedi, Ashok K.; Hardison, Sarah E.; Leopold Wager, Chrissy M.; Castro-Lopez, Natalia; Hole, Camaron R.; Wozniak, Karen L.; Wormley, Floyd L.

2017-01-01

Cryptococcosis is a fungal disease caused by multiple Cryptococcus serotypes; particularly C. neoformans (serotypes A and D) and C. gattii (serotypes B and C). To date, there is no clinically available vaccine to prevent cryptococcosis. Mice given an experimental pulmonary vaccination with a C. neoformans serotype A strain engineered to produce interferon-γ, denoted H99γ, are protected against a subsequent otherwise lethal experimental infection with C. neoformans serotype A. Thus, we determined the efficacy of immunization with C. neoformans strain H99γ to elicit broad-spectrum protection in BALB/c mice against multiple disparate Cryptococcus serotypes. We observed significantly increased survival rates and significantly decreased pulmonary fungal burden in H99γ immunized mice challenged with Cryptococcus serotypes A, B, or D compared to heat-killed H99γ (HKH99γ) immunized mice. Results indicated that prolonged protection against Cryptococcus serotypes B or D in H99γ immunized mice was CD4+ T cell dependent and associated with the induction of predominantly Th1-type cytokine responses. Interestingly, immunization with H99γ did not elicit greater protection against challenge with the Cryptococcus serotype C tested either due to low overall virulence of this strain or enhanced capacity of this strain to evade host immunity. Altogether, these studies provide “proof-of-concept” for the development of a cryptococcal vaccine that provides cross-protection against multiple disparate serotypes of Cryptococcus. PMID:29163469

Viral-Associated GN: Hepatitis C and HIV.

PubMed

Kupin, Warren L

2017-08-07

Viruses are capable of inducing a wide spectrum of glomerular disorders that can be categorized on the basis of the duration of active viremia: acute, subacute, or chronic. The variable responses of the adaptive immune system to each time period of viral infection results mechanistically in different histologic forms of glomerular injury. The unique presence of a chronic viremic carrier state with either hepatitis C (HCV) or HIV has led to the opportunity to study in detail various pathogenic mechanisms of viral-induced glomerular injury, including direct viral infection of renal tissue and the development of circulating immune complexes composed of viral antigens that deposit along the glomerular basement membrane. Epidemiologic data show that approximately 25%-30% of all HIV patients are coinfected with HCV and 5%-10% of all HCV patients are coinfected with HIV. This situation can often lead to a challenging differential diagnosis when glomerular disease occurs in this dual-infected population and requires the clinician to be familiar with the clinical presentation, laboratory workup, and pathophysiology behind the development of renal disease for both HCV and HIV. Both of these viruses can be categorized under the new classification of infection-associated GN as opposed to being listed as causes of postinfectious GN as has previously been applied to them. Neither of these viruses lead to renal injury after a latent period of controlled and inactive viremia. The geneses of HCV- and HIV-associated glomerular diseases share a total dependence on the presence of active viral replication to sustain renal injury so the renal disease cannot be listed under "postinfectious" GN. With the new availability of direct-acting antivirals for HCV and more effective combined antiretroviral therapy for HIV, successful remission and even regression of glomerular lesions can be achieved if initiated at an early stage. Copyright © 2017 by the American Society of Nephrology.

Herpes simplex virus serotype and entry receptor availability alter CNS disease in a mouse model of neonatal HSV.

PubMed

Kopp, Sarah J; Ranaivo, Hantamalala R; Wilcox, Douglas R; Karaba, Andrew H; Wainwright, Mark S; Muller, William J

2014-12-01

Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age. We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis. Mortality was attenuated in 7-d-old, wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 d after infection compared with adults and receptor-knockout newborns. Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

PubMed

Bosch, Irene; de Puig, Helena; Hiley, Megan; Carré-Camps, Marc; Perdomo-Celis, Federico; Narváez, Carlos F; Salgado, Doris M; Senthoor, Dewahar; O'Grady, Madeline; Phillips, Elizabeth; Durbin, Ann; Fandos, Diana; Miyazaki, Hikaru; Yen, Chun-Wan; Gélvez-Ramírez, Margarita; Warke, Rajas V; Ribeiro, Lucas S; Teixeira, Mauro M; Almeida, Roque P; Muñóz-Medina, José E; Ludert, Juan E; Nogueira, Mauricio L; Colombo, Tatiana E; Terzian, Ana C B; Bozza, Patricia T; Calheiros, Andrea S; Vieira, Yasmine R; Barbosa-Lima, Giselle; Vizzoni, Alexandre; Cerbino-Neto, José; Bozza, Fernando A; Souza, Thiago M L; Trugilho, Monique R O; de Filippis, Ana M B; de Sequeira, Patricia C; Marques, Ernesto T A; Magalhaes, Tereza; Díaz, Francisco J; Restrepo, Berta N; Marín, Katerine; Mattar, Salim; Olson, Daniel; Asturias, Edwin J; Lucera, Mark; Singla, Mohit; Medigeshi, Guruprasad R; de Bosch, Norma; Tam, Justina; Gómez-Márquez, Jose; Clavet, Charles; Villar, Luis; Hamad-Schifferli, Kimberly; Gehrke, Lee

2017-09-27

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks.

PubMed

Smoot, James C; Barbian, Kent D; Van Gompel, Jamie J; Smoot, Laura M; Chaussee, Michael S; Sylva, Gail L; Sturdevant, Daniel E; Ricklefs, Stacy M; Porcella, Stephen F; Parkins, Larye D; Beres, Stephen B; Campbell, David S; Smith, Todd M; Zhang, Qing; Kapur, Vivek; Daly, Judy A; Veasy, L George; Musser, James M

2002-04-02

Acute rheumatic fever (ARF), a sequelae of group A Streptococcus (GAS) infection, is the most common cause of preventable childhood heart disease worldwide. The molecular basis of ARF and the subsequent rheumatic heart disease are poorly understood. Serotype M18 GAS strains have been associated for decades with ARF outbreaks in the U.S. As a first step toward gaining new insight into ARF pathogenesis, we sequenced the genome of strain MGAS8232, a serotype M18 organism isolated from a patient with ARF. The genome is a circular chromosome of 1,895,017 bp, and it shares 1.7 Mb of closely related genetic material with strain SF370 (a sequenced serotype M1 strain). Strain MGAS8232 has 178 ORFs absent in SF370. Phages, phage-like elements, and insertion sequences are the major sources of variation between the genomes. The genomes of strain MGAS8232 and SF370 encode many of the same proven or putative virulence factors. Importantly, strain MGAS8232 has genes encoding many additional secreted proteins involved in human-GAS interactions, including streptococcal pyrogenic exotoxin A (scarlet fever toxin) and two uncharacterized pyrogenic exotoxin homologues, all phage-associated. DNA microarray analysis of 36 serotype M18 strains from diverse localities showed that most regions of variation were phages or phage-like elements. Two epidemics of ARF occurring 12 years apart in Salt Lake City, UT, were caused by serotype M18 strains that were genetically identical, or nearly so. Our analysis provides a critical foundation for accelerated research into ARF pathogenesis and a molecular framework to study the plasticity of GAS genomes.

Cryptococcosis Serotypes Impact Outcome and Provide Evidence of Cryptococcus neoformans Speciation.

PubMed

Desnos-Ollivier, Marie; Patel, Sweta; Raoux-Barbot, Dorothée; Heitman, Joseph; Dromer, Françoise

2015-06-09

Cryptococcus neoformans is a human opportunistic fungal pathogen causing severe disseminated meningoencephalitis, mostly in patients with cellular immune defects. This species is divided into three serotypes: A, D, and the AD hybrid. Our objectives were to compare population structures of serotype A and D clinical isolates and to assess whether infections with AD hybrids differ from infections with the other serotypes. For this purpose, we analyzed 483 isolates and the corresponding clinical data from 234 patients enrolled during the CryptoA/D study or the nationwide survey on cryptococcosis in France. Isolates were characterized in terms of ploidy, serotype, mating type, and genotype, utilizing flow cytometry, serotype- and mating type-specific PCR amplifications, and multilocus sequence typing (MLST) methods. Our results suggest that C. neoformans serotypes A and D have different routes of multiplication (primarily clonal expansion versus recombination events for serotype A and serotype D, respectively) and important genomic differences. Cryptococcosis includes a high proportion of proven or probable infections (21.5%) due to a mixture of genotypes, serotypes, and/or ploidies. Multivariate analysis showed that parameters independently associated with failure to achieve cerebrospinal fluid (CSF) sterilization by week 2 were a high serum antigen titer, the lack of flucytosine during induction therapy, and the occurrence of mixed infection, while infections caused by AD hybrids were more likely to be associated with CSF sterilization. Our study provides additional evidence for the possible speciation of C. neoformans var. neoformans and grubii and highlights the importance of careful characterization of causative isolates. Cryptococcus neoformans is an environmental fungus causing severe disease, estimated to be responsible for 600,000 deaths per year worldwide. This species is divided into serotypes A and D and an AD hybrid, and these could be considered two

Serotype 3 is a common serotype causing invasive pneumococcal disease in children less than 5 years old, as identified by real-time PCR.

PubMed

Selva, L; Ciruela, P; Esteva, C; de Sevilla, M F; Codina, G; Hernandez, S; Moraga, F; García-García, J J; Planes, A; Coll, F; Jordan, I; Cardeñosa, N; Batalla, J; Salleras, L; Dominguez, A; Muñoz-Almagro, C

2012-07-01

Serotype 3 is one of the most often detected pneumococcal serotypes in adults and it is associated with serious disease. In contrast, the isolation of serotype 3 by bacterial culture is unusual in children with invasive pneumococcal disease (IPD). The purpose of this study was to learn the serotype distribution of IPD, including culture-negative episodes, by using molecular methods in normal sterile samples. We studied all children<5 years of age with IPD admitted to two paediatric hospitals in Catalonia, Spain, from 2007 to 2009. A sequential real-time polymerase chain reaction (PCR) approach was added to routine methods for the detection and serotyping of pneumococcal infection. Among 257 episodes (219 pneumonia, 27 meningitis, six bacteraemia and five others), 33.5% were identified by culture and the rest, 66.5%, were detected exclusively by real-time PCR. The most common serotypes detected by culture were serotypes 1 (26.7%) and 19A (25.6%), and by real-time PCR, serotypes 1 (19.8%) and 3 (18.1%). Theoretical coverage rates by the PCV7, PCV10 and PCV13 vaccines were 10.5, 52.3 and 87.2%, respectively, for those episodes identified by culture, compared to 5.3, 31.6 and 60.2% for those identified only by real-time PCR. Multiplex real-time PCR has been shown to be useful for surveillance studies of IPD. Serotype 3 is underdiagnosed by culture and is important in paediatric IPD.

Immunological relationships between glucosyltransferases from Streptococcus mutans serotypes.

PubMed

Kuramitsu, H; Ingersoll, L

1976-09-01

Partially purified glycosyltransferase enzymes for Streptococcus mutans GS-5 (serotype c) have been utilized to prepare antibodies directed against the soluble glucan-synthesizing activity, GTF-B, and the insoluble-soluble glucan synthetic activity, GTF-A. Anti-GTF-A inhibited insoluble glucan formation catalyzed by the extracellular enzymes from strains GS-5 and FA-1 (serotype b) to a much greater extent than that of strains HS-6 (serotype a) or OMZ-176 (serotype d). This antibody fraction also inhibited both the cell-associated glucosyltransferase activities as well as the sucrose-mediated adherence of cells to glass surfaces by strains GS-5 and FA-1 but not that of strains HS-6 and OMZ-176. Anti-GTF-B inhibited soluble glucan formation catalyzed by the extracellular enzymes of strains GS-5 but not that of strain HS-6, FA-1, or OMZ-176. However, this antibody fraction did not strongly inhibit either the cell-associated glycosyltransferase activity or cellular adherence of any of the four strains. These results with body antibody fractions were also correlated with the ability of the antibodies to agglutinate the cells and form precipitin bands after immunodiffusion with the extracellular enzymes. Antibody prepared against the homogeneous soluble glucan-synthesizing enzyme demonstrated similar effects to the anti-GTF-B fraction. These results are discussed in terms of the antigenic relationships existing between the glucosyltransferases from different serotypes of S. mutans.

Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India.

PubMed

Rao, Arun Prasad; Austin, Ravi David

2014-07-01

The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years. Genomic DNA isolated from whole saliva of caries active (CA) and caries free (CF) groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR). Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes. Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80%) relative to CF group (43%). Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group. The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis.

Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

PubMed Central

Whitford, Margaret H.; Arruda, Valder R.; Stedman, Hansell H.; Kay, Mark A.; High, Katherine A.

2015-01-01

Abstract Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

PubMed

Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

2015-03-01

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

PubMed Central

White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

2013-01-01

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies.

PubMed

Scharinger, Eva J; Dietrich, Richard; Kleinsteuber, Ina; Märtlbauer, Erwin; Schauer, Kristina

2016-04-01

Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

PubMed

Brown, Nolan; Song, Liujiang; Kollu, Nageswara R; Hirsch, Matthew L

2017-06-01

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the

Evolutionary phylodynamics of foot-and-mouth disease virus serotypes O and A circulating in Vietnam.

PubMed

Le, Van Phan; Vu, Thi Thu Hang; Duong, Hong-Quan; Than, Van Thai; Song, Daesub

2016-11-29

Foot-and-mouth disease virus (FMDV) is one of the highest risk factors that affects the animal industry of the country. The virus causes production loss and high ratio mortality in young cloven-hoofed animals in Vietnam. The VP1 coding gene of 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) collected from endemic outbreaks during 2006-2014 were analyzed to investigate their phylogeny and genetic relationship with other available FMDVs globally. Phylogenetic analysis indicated that the serotype O strains were clustered into two distinct viral topotypes (the SEA and ME-SA), while the serotype A strains were all clustered into the genotype IX. Among the study strains, the amino acid sequence identities were shared at a level of 90.1-100, 92.9-100, and 92.8-100% for the topotypes SEA, ME-SA, and genotype IX, respectively. Substitutions leading to changes in the amino acid sequence, which are critical for the VP1 antigenic sites were also identified. Our results showed that the studied strains are most closely related to the recent FMDV isolates from Southeast Asian countries (Myanmar, Thailand, Cambodia, Malaysia, and Laos), but are distinct from the earlier FMDV isolates within the genotypes. This study provides important evidence of recent movement of FMDVs serotype O and A into Vietnam within the last decade and their genetic accumulation to be closely related to strains causing FMD in surrounding countries.

Uptake of reovirus serotype 1 by the lungs from the bloodstream is mediated by the viral hemagglutinin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verdin, E.M.; Lynn, S.P.; Fields, B.N.

1988-02-01

The authors used the mammalian reoviruses to determine the molecular basis of the clearance of a virus from the bloodstream by specific organs. Reovirus serotypes 1 (T1) and 3 (T3) were radiolabeled with ({sup 35}S)methionine or {sup 125}I, and the viruses were injected intravenously into weanling rats. The distribution of radioactivity within the animals was determined at various times after the injection. Both viruses were cleared rapidly from the bloodstream and concentrated in different organs. Reovirus T1 was found predominantly in the lungs and liver, whereas T3 was found predominantly in the liver, with very little virus in the lungs.more » Using intertypic reassortants, they determined that the T1 S1 gene, which encodes the viral hemagglutinin ({sigma}1 protein), is responsible for the difference in uptake of T1 and T3 by the lungs. The genetic mapping was extended by using several approaches. (i) T1 subjected to limited proteolytic digestion with chymotrypsin was cleared efficiently by the lungs despite the removal of {sigma}3 and digestion of {mu}1C to {delta}. (ii) Uptake of T1 by the lungs was totally inhibited by incubation of T1 with an anti-{sigma}1 monoclonal antibody or its Fab fragment before injection. (iii) A reovirus T1 variant in the {sigma}1 protein was poorly taken up by the lungs. These data indicate that clearance of reovirus from the bloodstream by the lungs is dependent on the presence of the T1 or {sigma}1 protein.« less

Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.

PubMed

Lalwani, A K; Walsh, B J; Reilly, P G; Muzyczka, N; Mhatre, A N

1996-07-01

Gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. However, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. The purpose of this study was to assess the feasibility of introducing genetic material directly into the peripheral auditory system using adeno-associated virus (AAV) as the transfection vector and Hartley guinea pigs as the animal model. Approximately 10(5) particles of AAV containing the bacterial beta-galactosidase (beta-gal) sequence with Ad 2 major late promoter were infused into the cochlea of the animal with the aid of an osmotic minipump. Animals were killed after 2 weeks. Two Hartley guinea pigs with intracochlear saline infusion and four unoperated (nonperfused) animals served as negative controls. Both, the infused and the contralateral, non-infused cochleae were harvested from each animal, decalcified, and embedded in paraffin. Sections, 8 microns in width, were cut from the embedded cochleae and assayed for beta-gal expression via immunohistochemistry. Animals perfused with AAV showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti in the perfused cochlea and a much weaker staining but with similar pattern in the contralateral ear. Cochleae from saline-infused and unoperated animals were devoid of the DAB stain. This study demonstrates for the first time in vivo expression of a foreign gene within the mammalian inner ear resulting from its localized, AAV-mediated introduction. The ability to introduce and stably express exogenous genetic material in the peripheral auditory system will have both experimental and therapeutic benefits. These results lay the groundwork for future studies assessing the

Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

PubMed

Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

2010-09-01

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

siRNAs encapsulated in recombinant capsid protein derived from Dengue serotype 2 virus inhibits the four serotypes of the virus and proliferation of cancer cells.

PubMed

Kumar, A S Manoj; Reddy, G E C Vidyadhar; Rajmane, Yogesh; Nair, Soumya; Pai Kamath, Sangita; Sreejesh, Greeshma; Basha, Khalander; Chile, Shailaja; Ray, Kriti; Nelly, Vivant; Khadpe, Nilesh; Kasturi, Ravishankar; Ramana, Venkata

2015-01-10

siRNA delivery potential of the Dengue virus capsid protein in cultured cells was recently reported, but target knockdown potential in the context of specific diseases has not been explored. In this study we have evaluated the utility of the protein as an siRNA carrier for anti Dengue viral and anti cancer applications using cell culture systems. We show that target specific siRNAs delivered using the capsid protein inhibit infection by the four serotypes of Dengue virus and proliferation of two cancer cell lines. Our data confirm the potential of the capsid for anti Dengue viral and anti cancer RNAi applications. In addition, we have optimized a fermentation strategy to improve the yield of Escherichia coli expressed D2C protein since the reported yields of E. coli expressed flaviviral capsid proteins are low. Copyright © 2014 Elsevier B.V. All rights reserved.

Serotype specific polymerase chain reaction identifies a higher prevalence of streptococcus mutans serotype k and e in a random group of children with dental caries from the Southern region of India

PubMed Central

Rao, Arun Prasad; Austin, Ravi David

2014-01-01

Background: The development of dental caries has been associated with the oral prevalence of Streptococcus mutans. Four serotypes of S. mutans have been reported, namely serotype c, e, f, and k that are classified based on the composition and linkages of cell wall polysaccharides, response to physiological reactions, sero-specificity and 16s rRNA homology. Although the oral prevalence of S. mutans serotype c in Indian subjects with or without caries is known, the prevalence of the other three serotypes, e, f, and k are not known. Hence in this study, we have investigated the occurrence of the e, f, and k serotypes in children with or without caries within the age group of 6-12 years. Materials and Methods: Genomic DNA isolated from whole saliva of caries active (CA) and caries free (CF) groups were first screened for the presence of S. mutans by strain specific polymerase chain reaction (PCR). Those samples that tested positive for the presence of S. mutans were further analyzed by serotype specific PCR to identify the prevalence of the serotypes. Results: Strain specific PCR indicated a higher prevalence of S. mutans in CA group (80%) relative to CF group (43%). Further analysis of the S. mutans positive samples in both groups indicated a higher prevalence of serotype k and e, followed by serotype f in CA group. Conclusion: The present data clearly establishes a novel S. mutans serotype prevalence hierarchy in children from this region, compared with those that have been reported elsewhere. Besides, the data are also clinically significant as the occurrence of serotype k has been associated with infective endocarditis. PMID:25191062

Transmission dynamics of two dengue serotypes with vaccination scenarios.

PubMed

González Morales, N L; Núñez-López, M; Ramos-Castañeda, J; Velasco-Hernández, J X

2017-05-01

In this work we present a mathematical model that incorporates two Dengue serotypes. The model has been constructed to study both the epidemiological trends of the disease and conditions that allow coexistence in competing strains under vaccination. We consider two viral strains and temporary cross-immunity with one vector mosquito population. Results suggest that vaccination scenarios will not only reduce disease incidence but will also modify the transmission dynamics. Indeed, vaccination and cross immunity period are seen to decrease the frequency and magnitude of outbreaks but in a differentiated manner with specific effects depending upon the interaction vaccine and strain type. Copyright © 2016 Elsevier Inc. All rights reserved.

[Meningitis outbreak caused by Echovirus serotype 30 in the Valencian Community].

PubMed

Juliá, M Lirios; Colomina, Javier; Domínguez, Victoria; Orta, Nieves; Guerrero, Antonio

2009-05-01

Aseptic meningitis can be caused by several agents, and in many cases the etiology remains unknown. The aim of this study to analyze the clinical and epidemiological characteristics of a meningitis outbreak detected in Health Department 11 of the Valencian Community (Spain). The study was performed in children hospitalized between November and December 2006 with meningitis symptoms, CSF pleocytosis, and negative CSF bacteriological culture. An epidemiological survey was conducted among cases and family members. Virus detection and phylogenetic analysis were performed with molecular biology techniques. The outbreak affected at least 44 children, with a mean age (standard deviation) of 5.5 years (2.9). The average hospital stay was 3.1 days and outcome was favorable in all cases. In 24 patients the CSF specimen sufficed for viral detection by PCR; enteroviruses ultimately serotyped as echovirus 30 were detected in 12 of them (50%). This serotype has been recently found in other parts of our country. Detection of echovirus 30 in CSF and the epidemiological presentation of cases enabled determination of the etiology of the outbreak. This finding coincided in time with other outbreaks of echovirus 30 in Spain, a fact that may explain the epidemic situation in the Valencian Community during 2006. Establishment of a national surveillance network for monitoring systemic enterovirus infection would provide data on the circulation patterns and identify new emerging serotypes.

Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease

PubMed Central

Karumuthil-Melethil, Subha; Kalburgi, Sahana Nagabhushan; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D.; Keimel, John G.; Mark, Brian L.; Mahuran, Don; Walia, Jagdeep S.; Gray, Steven J.

2016-01-01

GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α–β), “A” isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP– GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter–intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the

Molecular detection assay of five Salmonella serotypes of public interest: Typhimurium, Enteritidis, Newport, Heidelberg, and Hadar.

PubMed

Bugarel, M; Tudor, A; Loneragan, G H; Nightingale, K K

2017-03-01

Foodborne illnesses due to Salmonella represent an important public-health concern worldwide. In the United States, a majority of Salmonella infections are associated with a small number of serotypes. Furthermore, some serotypes that are overrepresented among human disease are also associated with multi-drug resistance phenotypes. Rapid detection of serotypes of public-health concern might help reduce the burden of salmonellosis cases and limit exposure to multi-drug resistant Salmonella. We developed a two-step real-time PCR-based rapid method for the identification and detection of five Salmonella serotypes that are either overrepresented in human disease or frequently associated with multi-drug resistance, including serotypes Enteritidis, Typhimurium, Newport, Hadar, and Heidelberg. Two sets of four markers were developed to detect and differentiate the five serotypes. The first set of markers was developed as a screening step to detect the five serotypes; whereas, the second set was used to further distinguish serotypes Heidelberg, Newport and Hadar. The utilization of these markers on a two-step investigation strategy provides a diagnostic specificity of 97% for the detection of Typhimurium, Enteritidis, Heidelberg, Infantis, Newport and Hadar. The diagnostic sensitivity of the detection makers is >96%. The availability of this two-step rapid method will facilitate specific detection of Salmonella serotypes that contribute to a significant proportion of human disease and carry antimicrobial resistance. Published by Elsevier B.V.

Intrajugular Vein Delivery of AAV9-RNAi Prevents Neuropathological Changes and Weight Loss in Huntington's Disease Mice

PubMed Central

Dufour, Brett D; Smith, Catherine A; Clark, Randall L; Walker, Timothy R; McBride, Jodi L

2014-01-01

Huntington's disease (HD) is a fatal neurological disorder caused by a CAG repeat expansion in the HTT gene, which encodes a mutant huntingtin protein (mHTT). The mutation confers a toxic gain of function on huntingtin, leading to widespread neurodegeneration and inclusion formation in many brain regions. Although the hallmark symptom of HD is hyperkinesia stemming from striatal degeneration, several other brain regions are affected which cause psychiatric, cognitive, and metabolic symptoms. Additionally, mHTT expression in peripheral tissue is associated with skeletal muscle atrophy, cardiac failure, weight loss, and diabetes. We, and others, have demonstrated a prevention of motor symptoms in HD mice following direct striatal injection of adeno-associated viral vector (AAV) serotype 1 encoding an RNA interference (RNAi) construct targeting mutant HTT mRNA (mHTT). Here, we expand these efforts and demonstrate that an intrajugular vein injection of AAV serotype 9 (AAV9) expressing a mutant HTT-specific RNAi construct significantly reduced mHTT expression in multiple brain regions and peripheral tissues affected in HD. Correspondingly, this approach prevented atrophy and inclusion formation in key brain regions as well as the severe weight loss germane to HD transgenic mice. These results demonstrate that systemic delivery of AAV9-RNAi may provide more widespread clinical benefit for patients suffering from HD. PMID:24390280

Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis

PubMed Central

Sévellec, Yann; Vignaud, Marie-Léone; Granier, Sophie A.; Lailler, Renaud; Feurer, Carole; Le Hello, Simon; Mistou, Michel-Yves; Cadel-Six, Sabrina

2018-01-01

In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours.

PubMed

Buhles, Alexandra; Collins, Sara A; van Pijkeren, Jan P; Rajendran, Simon; Miles, Michelle; O'Sullivan, Gerald C; O'Hanlon, Deirdre M; Tangney, Mark

2009-03-09

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metastasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate

Myanmar Dengue Outbreak Associated with Displacement of Serotypes 2, 3, and 4 by Dengue 1

PubMed Central

Thu, Hlaing Myat; Lowry, Kym; Myint, Thein Thein; Shwe, Than Nu; Han, Aye Maung; Khin, Kyu Kyu; Thant, Kyaw Zin; Thein, Soe

2004-01-01

In 2001, Myanmar (Burma) had its largest outbreak of dengue—15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses. PMID:15200847

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

PubMed

Cunningham, Sharon C; Siew, Susan M; Hallwirth, Claus V; Bolitho, Christine; Sasaki, Natsuki; Garg, Gagan; Michael, Iacovos P; Hetherington, Nicola A; Carpenter, Kevin; de Alencastro, Gustavo; Nagy, Andras; Alexander, Ian E

2015-08-01

Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. © 2015 by

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

PubMed

Parzych, Elizabeth M; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M; High, Katherine A; Levine, Matthew H; Ertl, Hildegund C J

2013-04-01

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Effects of Immunosuppression on Circulating Adeno-Associated Virus Capsid-Specific T cells in Humans

PubMed Central

Parzych, Elizabeth M.; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M.; High, Katherine A.; Levine, Matthew H.

2013-01-01

Abstract In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4+ T cells, whereas numbers of circulating CD8+ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients. PMID:23461589

Haemophilus influenzae serotype a meningitis.

PubMed

de Pádua, Rubia Andreia Falleiros; de Lima Scodro, Regiane Bertin; Ghiraldi, Luciana Dias; Siqueira, Vera Lúcia Dias; Yamashita, Yandara Keiko; Helbel, César; Cardoso, Rosilene Fressatti

2009-01-01

This work describes a case of Haemophilus influenzae serotype a meningitis in Brazil, after almost a decade since the introduction of Haemophilus influenzae serotype b conjugate vaccine. Uncertainty about the replacement of H. influenzae serotypes as a cause of invasive diseases justifies continuous surveillance, coupled with investigations of carriage rates and requirements of chemoprophylaxis in contact persons.

Transplacental infection in goats experimentally infected with a European strain of bluetongue virus serotype 8.

PubMed

Coetzee, Peter; Stokstad, Maria; Myrmel, Mette; Mutowembwa, Paidamwoyo; Loken, Torleiv; Venter, Estelle H; Van Vuuren, Moritz

2013-08-01

The capability of the recently emerged European strain of bluetongue virus serotype 8 (BTV-8) to cross the ruminant placenta has been established in experimental and field studies in both sheep and cattle. Seroprevalence rates in goats in North-Western Europe were high during the recent outbreak of BTV-8; however the capability of the virus to infect goats through the transplacental route has not been established. In the present study, four Saanen goats were inoculated with the European strain of BTV-8 at 62 days of gestation; this resulted in mild clinical signs, however gross lesions observed post mortem were more severe. Viral RNA was detected by real-time RT-PCR in blood and tissue samples from three fetuses harvested from two goats at 43 days post infection. Conventional RT-PCR and genome sequencing targeting viral segment 2 confirmed infection of brain tissue with BTV-8 in two of these fetuses. In total, five of six fetuses demonstrated lesions that may have been associated with transplacental infection with BTV. Infected fetuses did not demonstrate neurological lesions. Low viral RNA concentrations in fetal blood and tissue further suggest that the infected fetuses would probably not have been born viraemic. The implications of these findings with regards to the epidemiology and overwintering of BTV-8 in Europe remains unclear. Copyright © 2013 Elsevier Ltd. All rights reserved.

Neurological diseases associated with viral and Mycoplasma pneumoniae infections

PubMed Central

Assaad, F.; Gispen, R.; Kleemola, M.; Syrůček, L.; Esteves, K.

1980-01-01

In 1963 the World Health Organization established a system for the collection and dissemination of information on viral infections and by 1976, laboratories in 49 countries were participating in this scheme. The present study is in two parts: part 1 is an analysis of almost 60 000 reports on neurological disease associated with viral and Mycoplasma pneumoniae infections reported during the 10-year period 1967-76. This analysis showed a steady increase in the yearly number of reports of viral neurological diseases, which closely followed the general increase in the overall reporting of virus diseases. Likewise, the seasonal pattern was similar to that seen in general for any given virus. Over 75% of the cases were in children. Over half of all viral neurological diseases were associated with enteroviruses, while the myxoviruses accounted for almost 30%. Among the myxoviruses, mumps virus was by far the most frequently reported. The polioviruses were the agents most commonly detected in cases of paralytic disease. The other enteroviruses, mumps virus, and the herpesviruses were the most frequently reported viruses in cases of aseptic meningitis or encephalitis. On the other hand, one-third to over one-half of the reports on the myxoviruses (excluding mumps and measles) related to ill-defined clinical conditions. Part 2 of the study deals in particular with viruses whose role in neurological disease is less well documented. One laboratory reported an outbreak of adenoviral aseptic meningitis in Czechoslovakia, while another described neurological disease associated with M. pneumoniae infection in Finland. Part 2 also includes a detailed appraisal of viral infections diagnosed in the Netherlands during the period 1973-76. The results are very similar to those routinely reported. PMID:6249511

AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

PubMed

Kiyota, T; Ingraham, K L; Swan, R J; Jacobsen, M T; Andrews, S J; Ikezu, T

2012-07-01

Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

Genetic associations with viral respiratory illnesses and asthma control in children

PubMed Central

Loisel, Dagan A; Du, Gaixin; Ahluwalia, Tarunveer Singh; Tisler, Christopher J.; Evans, Michael D.; Myers, Rachel A.; Gangnon, Ronald E.; Kreiner-Møller, Eskil; Bønnelykke, Klaus; Bisgaard, Hans; Jackson, Daniel J.; Lemanske, Robert F.; Nicolae, Dan L.; Gern, James E.; Ober, Carole

2015-01-01

Background Viral respiratory infections can cause acute wheezing illnesses in children and exacerbations of asthma. Objective We sought to identify variation in genes with known antiviral and pro-inflammatory functions to identify specific associations with more severe viral respiratory illnesses and the risk of virus-induced exacerbations during the peak fall season. Methods The associations between genetic variation at 326 SNPs in 63 candidate genes and 10 phenotypes related to viral respiratory infection and asthma control were examined in 226 children enrolled in the RhinoGen study. Replication of asthma control phenotypes was performed in 2,128 children in the Copenhagen Prospective Study on Asthma in Childhood (COPSAC). Significant associations in RhinoGen were further validated using virus-induced wheezing illness and asthma phenotypes in an independent sample of 122 children enrolled in the Childhood Origins of Asthma birth cohort study (COAST). Results A significant excess of P values smaller than 0.05 was observed in the analysis of the 10 RhinoGen phenotypes. Polymorphisms in 12 genes were significantly associated with variation in the four phenotypes showing a significant enrichment of small P values. Six of those genes (STAT4, JAK2, MX1, VDR, DDX58, and EIF2AK2) also showed significant associations with asthma exacerbations in the COPSAC study or with asthma or virus-induced wheezing phenotypes in the COAST study. Conclusions We identified genetic factors contributing to individual differences in childhood viral respiratory illnesses and virus-induced exacerbations of asthma. Defining mechanisms of these associations may provide insight into the pathogenesis of viral respiratory infections and virus-induced exacerbations of asthma. PMID:26399222

Intranasal Adeno-Associated Virus Mediated Gene Delivery and Expression of Human Iduronidase in the Central Nervous System: A Noninvasive and Effective Approach for Prevention of Neurologic Disease in Mucopolysaccharidosis Type I.

PubMed

Belur, Lalitha R; Temme, Alexa; Podetz-Pedersen, Kelly M; Riedl, Maureen; Vulchanova, Lucy; Robinson, Nicholas; Hanson, Leah R; Kozarsky, Karen F; Orchard, Paul J; Frey, William H; Low, Walter C; McIvor, R Scott

2017-07-01

Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.

Virally delivered Channelrhodopsin-2 Safely and Effectively Restores Visual Function in Multiple Mouse Models of Blindness

PubMed Central

Doroudchi, M Mehdi; Greenberg, Kenneth P; Liu, Jianwen; Silka, Kimberly A; Boyden, Edward S; Lockridge, Jennifer A; Arman, A Cyrus; Janani, Ramesh; Boye, Shannon E; Boye, Sanford L; Gordon, Gabriel M; Matteo, Benjamin C; Sampath, Alapakkam P; Hauswirth, William W; Horsager, Alan

2011-01-01

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness. PMID:21505421

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

PubMed

Nambiar, Bindu; Cornell Sookdeo, Cathleen; Berthelette, Patricia; Jackson, Robert; Piraino, Susan; Burnham, Brenda; Nass, Shelley; Souza, David; O'Riordan, Catherine R; Vincent, Karen A; Cheng, Seng H; Armentano, Donna; Kyostio-Moore, Sirkka

2017-02-01

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Serotype and mating type characterization of Cryptococcus neoformans by multiplex PCR.

PubMed

Carvalho, Vívian Gonçalves; Terceti, Mateus Souza; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues; Lyon, Juliana Pereira; de Siqueira, Antônio Martins; Franco, Marília Caixeta

2007-01-01

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (A alpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

El Niño-Southern Oscillation, local weather and occurrences of dengue virus serotypes

NASA Astrophysics Data System (ADS)

Huang, Xiaodong; Clements, Archie C. A.; Williams, Gail; Devine, Gregor; Tong, Shilu; Hu, Wenbiao

2015-11-01

Severe dengue fever is usually associated with secondary infection by a dengue virus (DENV) serotype (1 to 4) that is different to the serotype of the primary infection. Dengue outbreaks only occur following importations of DENV in Cairns, Australia. However, the majority of imported cases do not result in autochthonous transmission in Cairns. Although DENV transmission is strongly associated with the El Niño-Southern Oscillation (ENSO) climate cycle and local weather conditions, the frequency and potential risk factors of infections with the different DENV serotypes, including whether or not they differ, is unknown. This study used a classification tree model to identify the hierarchical interactions between Southern Oscillation Index (SOI), local weather factors, the presence of imported serotypes and the occurrence of the four autochthonous DENV serotypes from January 2000-December 2009 in Cairns. We found that the 12-week moving average of SOI and the 2-week moving average of maximum temperature were the most important factors influencing the variation in the weekly occurrence of the four DENV serotypes, the likelihoods of the occurrence of the four DENV serotypes may be unequal under the same environmental conditions, and occurrence may be influenced by changes in global and local environmental conditions in Cairns.

Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons

PubMed Central

Morral, Núria; O’Neal, Wanda; Rice, Karen; Leland, Michele; Kaplan, Johanne; Piedra, Pedro A.; Zhou, Heshan; Parks, Robin J.; Velji, Rizwan; Aguilar-Córdova, Estuardo; Wadsworth, Samuel; Graham, Frank L.; Kochanek, Stefan; Carey, K. Dee; Beaudet, Arthur L.

1999-01-01

The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration of transgene expression, which can be related to immune responses and to toxic effects of viral proteins. In addition, readministration is usually ineffective unless the animals are immunocompromised or a different adenovirus serotype is used. Recently, adenoviral vectors devoid of all viral coding sequences (helper-dependent or gutless vectors) have been developed to avoid expression of viral proteins. In mice, liver-directed gene transfer with AdSTK109, a helper-dependent adenoviral (Ad) vector containing the human α1-antitrypsin (hAAT) gene, resulted in sustained expression for longer than 10 months with negligible toxicity to the liver. In the present report, we have examined the duration of expression of AdSTK109 in the liver of baboons and compared it to first-generation vectors expressing hAAT. Transgene expression was limited to approximately 3–5 months with the first-generation vectors. In contrast, administration of AdSTK109 resulted in transgene expression for longer than a year in two of three baboons. We have also investigated the feasibility of circumventing the humoral response to the virus by sequential administration of vectors of different serotypes. We found that the ineffectiveness of readministration due to the humoral response to an Ad5 first-generation vector was overcome by use of an Ad2-based vector expressing hAAT. These data suggest that long-term expression of transgenes should be possible by combining the reduced immunogenicity and toxicity of helper-dependent vectors with sequential delivery of vectors of different serotypes. PMID:10536005

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

PubMed Central

Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

2015-01-01

Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency. PMID:26305933

Emerging Opportunities for Serotypes of Botulinum Neurotoxins

PubMed Central

Peng Chen, Zhongxing; Morris, J. Glenn; Rodriguez, Ramon L.; Shukla, Aparna Wagle; Tapia-Núñez, John; Okun, Michael S.

2012-01-01

Background: Two decades ago, botulinum neurotoxin (BoNT) type A was introduced to the commercial market. Subsequently, the toxin was approved by the FDA to address several neurological syndromes, involving muscle, nerve, and gland hyperactivity. These syndromes have typically been associated with abnormalities in cholinergic transmission. Despite the multiplicity of botulinal serotypes (designated as types A through G), therapeutic preparations are currently only available for BoNT types A and B. However, other BoNT serotypes are under study for possible clinical use and new clinical indications; Objective: To review the current research on botulinum neurotoxin serotypes A-G, and to analyze potential applications within basic science and clinical settings; Conclusions: The increasing understanding of botulinal neurotoxin pathophysiology, including the neurotoxin’s effects on specific neuronal populations, will help us in tailoring treatments for specific diagnoses, symptoms and patients. Scientists and clinicians should be aware of the full range of available data involving neurotoxin subtypes A-G. PMID:23202312

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Isolates from Pediatric Patients in Singapore

PubMed Central

Soh, Shirlena Wee-Ling; Poh, Chit Laa; Lin, Raymond V. Tzer Pin

2000-01-01

One hundred eighty Streptococcus pneumoniae strains isolated from children at a pediatric hospital in Singapore from 1997 to 1999 were serotyped and their antimicrobial susceptibility patterns were determined. Sixty-three percent of the isolates were resistant to penicillin. Significantly large numbers of the strains investigated were resistant to trimethoprim-sulfamethoxazole (87.8%), tetracycline (71.7%), erythromycin (67.8%), and chloramphenicol (40%). Penicillin and multidrug resistance was mostly associated with the frequently isolated S. pneumoniae isolates of serotypes (serotypes 19F, 23F, 6B, and 14). Isolates of serotype 19F, the serotype most commonly encountered in Singapore (41.1%), had the highest prevalence of penicillin (78.4%) and multidrug resistance (94.6%). Most of the invasive S. pneumoniae isolates (8 of 17; 47.1%) were of serotype 14. PMID:10898701

Identification of Haemophilus influenzae clones associated with invasive disease a decade after introduction of H. influenzae serotype b vaccination in Italy.

PubMed

Giufrè, Maria; Cardines, Rita; Accogli, Marisa; Pardini, Manuela; Cerquetti, Marina

2013-08-01

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla(TEM) gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were β-lactamase positive, harboring the bla(TEM) gene. Most bla(TEM) genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the β-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.

Simultaneous detection and serotyping of dengue infection using single tube multiplex CDC Dengue Real-Time RT-PCR from India.

PubMed

Sharma, Shashi; Tandel, Kundan; Danwe, Surabhi; Bhatt, Puneet; Dash, P K; Ranjan, Praveer; Rathi, K R; Gupta, Rajiv Mohan; Parida, M M

2018-03-01

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well

Serotyping of Streptococcus pneumoniae Based on Capsular Genes Polymorphisms

PubMed Central

Raymond, Frédéric; Boucher, Nancy; Allary, Robin; Robitaille, Lynda; Lefebvre, Brigitte; Tremblay, Cécile

2013-01-01

Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. A novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Autolysin, pneumolysin and eight genes located in the capsular operon were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system to identify capsular serotypes. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. We show that typing only 12 polymorphisms located in the capsular operon allows the identification at the serotype level of 22 serotypes and the assignation of 24 other serotypes to a subgroup of serotypes. Overall, 126 samples (75.9%) were correctly serotyped, 14 were assigned to a member of the same serogroup, 8 rare serotypes were erroneously serotyped, and 18 gave negative serotyping results. Most of the discrepancies involved rare serotypes or serotypes that are difficult to discriminate using a DNA-based approach, for example 6A and 6B. The assay was also tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. Overall, 89% of specimens positive for pneumolysin were serotyped, demonstrating that this method does not require culture to serotype clinical specimens. The assay showed no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

PubMed Central

Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

2004-01-01

Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

PubMed Central

Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard

2015-01-01

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented. PMID:26527147

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-04-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

PubMed Central

Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

1985-01-01

Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains. Images PMID:3980081

First environmental isolation of Cryptococcus gattii serotype B, from Cúcuta, Colombia.

PubMed

Firacative, Carolina; Torres, Germán; Rodríguez, María Claudia; Escandón, Patricia

2011-03-01

In Cúcuta, Cryptococcus gattii serotype B is commonly recovered from immunocompetent patients with cryptococcosis, but it has not been recovered from the environment in spite of its high incidence which is 77% out of reported cases. The aim of this work was to carry out an extensive environmental sampling in Cúcuta, in an attempt to isolate C. gattii serotype B and to expand our knowledge about the ecology and epidemiology of this important yeast. Samples associated with 3,634 trees from 40 zones of Cúcuta were collected and processed with 28 samples collected near the houses of four patients with cryptococcosis caused by C. gattii serotype B. The serotype of the recovered isolates was done using multiplex PCR, molecular patterns were determined by RFLP of the URA5 gene and mating type was determined using the primers MfαU, MfαL, MFa2U and MFa2L. In total, 4,389 samples were processed and one isolate of C. gattii serotype B (VGI/a), two isolates of C. gattii serotype C (VGIII/α) and three isolates of C. neoformans var. grubii, serotype A (VNI/α), were recovered. The density of the recovered isolates varied from 50 to 350 cfu/g of soil. This is the first report on the environmental isolation of C. gattii serotype B from Cúcuta. However, because of the low rate of recovery of isolates from soil only, the environmental niche of C. gattii has not been established and further environmental studies in Cúcuta are necessary, owing that this serotype is not only causing cryptococcosis but also has shown a higher virulence after the Vancouver outbreak.

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses

PubMed Central

Shigetomi, Eiji; Bushong, Eric A.; Haustein, Martin D.; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V.; Ellisman, Mark H.

2013-01-01

Intracellular Ca2+ transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca2+ has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca2+ indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca2+ signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca2+ microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca2+ signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function. PMID:23589582

Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

PubMed

Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V; Ellisman, Mark H; Khakh, Baljit S

2013-05-01

Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

PubMed

Tadokoro, Takahiro; Miyanohara, Atsushi; Navarro, Michael; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Marsala, Silvia; Platoshyn, Oleksandr; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Lukacova, Nada; Bimbova, Katarina; Marsala, Martin

2017-07-13

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10 13 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

PubMed

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty

Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo

PubMed Central

Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

2018-01-01

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP−/−) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV

Viral blips during suppressive antiretroviral treatment are associated with high baseline HIV-1 RNA levels.

PubMed

Sörstedt, Erik; Nilsson, Staffan; Blaxhult, Anders; Gisslén, Magnus; Flamholc, Leo; Sönnerborg, Anders; Yilmaz, Aylin

2016-06-21

Many HIV-1-infected patients on suppressive antiretroviral therapy (ART) have transiently elevated HIV RNA levels. The clinical significance of these viral blips is uncertain. We have determined the incidence of blips and investigated important associations in the Swedish HIV-cohort. HIV-1-infected ART naïve adults who commenced ART 2007-2013 were retrospectively included. Viral blips were defined as a transient viral load between 50 and 500 copies/mL Subjects not suppressed after six months on ART were excluded. Viral blips were found in 76/735 included subjects (10.3 %) and in 90/4449 samples (2.0 %). Median blip viral load was 76 copies/mL (range 56-138). Median follow-up time was 170 weeks (range 97-240). Baseline viral load was higher in subjects with viral blips (median log10 4.85 copies/mL) compared with subjects without blips (median log10 4.55 copies/mL) (p < 0.01). There was a significant association between viral blips and risk for subsequent virological failure (p < 0.001). The Swedish national HIV-cohort has a low incidence of viral blips (10 %). Blips were associated with high baseline viral load and an increased risk of subsequent virological failure.

Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India.

PubMed

Reddy, Y V; Susmitha, B; Patil, S; Krishnajyothi, Y; Putty, K; Ramakrishna, K V; Sunitha, G; Devi, B V; Kavitha, K; Deepthi, B; Krovvidi, S; Reddy, Y N; Reddy, G H; Singh, K P; Maan, N S; Hemadri, D; Maan, S; Mertens, P P; Hegde, N R; Rao, P P

2018-04-01

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population. © 2017 Blackwell Verlag GmbH.

Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laughlin, C.A.; Myers, M.W.; Risin, D.L.

1979-04-15

We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious butmore » interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.« less

Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes

PubMed Central

2015-01-01

Background It is known that mRNA folding can affect and regulate various gene expression steps both in living organisms and in viruses. Previous studies have recognized functional RNA structures in the genome of the Dengue virus. However, these studies usually focused either on the viral untranslated regions or on very specific and limited regions at the beginning of the coding sequences, in a limited number of strains, and without considering evolutionary selection. Results Here we performed the first large scale comprehensive genomics analysis of selection for local mRNA folding strength in the Dengue virus coding sequences, based on a total of 1,670 genomes and 4 serotypes. Our analysis identified clusters of positions along the coding regions that may undergo a conserved evolutionary selection for strong or weak local folding maintained across different viral variants. Specifically, 53-66 clusters for strong folding and 49-73 clusters for weak folding (depending on serotype) aggregated of positions with a significant conservation of folding energy signals (related to partially overlapping local genomic regions) were recognized. In addition, up to 7% of these positions were found to be conserved in more than 90% of the viral genomes. Although some of the identified positions undergo frequent synonymous / non-synonymous substitutions, the selection for folding strength therein is preserved, and thus cannot be trivially explained based on sequence conservation alone. Conclusions The fact that many of the positions with significant folding related signals are conserved among different Dengue variants suggests that a better understanding of the mRNA structures in the corresponding regions may promote the development of prospective anti- Dengue vaccination strategies. The comparative genomics approach described here can be employed in the future for detecting functional regions in other pathogens with very high mutations rates. PMID:26449467

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8.

PubMed

Vandaele, Leen; Wesselingh, Wendy; De Clercq, Kris; De Leeuw, Ilse; Favoreel, Herman; Van Soom, Ann; Nauwynck, Hans

2011-01-24

Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 10(3.8) or 10(4.9) TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.

Serotype diversity of Streptococcus mutans and caries activity in children in Argentina.

PubMed

Carletto-Körber, F P; González-Ittig, R E; Jimenez, M G; Cornejo, L S

2015-09-01

The purpose of this study was to analyse the serotype distribution of S. mutans and their association with caries activity in school children from Córdoba, Argentina. Clinical examination was performed in 133 children. The dmft+DMFT and Significant Caries (SiC) indices were calculated to identify individuals with high caries activity. After DNA extractions of S. mutans strains, serotypes were determined by PCR amplifications. The median caries activity of each serotype group was compared using a non-parametric Kruskall-Wallis test. We obtained S. mutans strains from stimulated saliva of 94 children. The mean dmft+DMFT was 4.14 and the mean SiC index was 8.65. Serotype c was the most frequent (53.2%), followed by e (31.9%), f (8.5%) and k (6.4%). The comparison between the SiC and Non-Sic groups showed significant differences in the frequency of serotypes c and k. The median caries activity was non-significant in the different serotypes. The difference between the serotype frequencies detected in Argentina compared to those of other countries could be related with contrasting dietary habits. The results obtained in the present study would increase the knowledge about the epidemiology of dental caries in children from Argentina.

Hemorrhagic colitis associated with Salmonella enterica serotype Infantis infection in a captive western lowland gorilla (Gorilla gorilla gorilla) in Brazil.

PubMed

Paixão, Tatiane A; Malta, Marcelo C C; Soave, Semíramis A; Tinoco, Herlandes P; Costa, Maria E L T; Pessanha, Angela T; Silva, Rodrigo O S; Coura, Fernanda M; Costa, Luciana F; Turchetti, Andreia P; Lobato, Francisco C F; Melo, Marilia M; Heinemann, Marcos B; Santos, Renato L

2014-04-01

Enteric diseases are among the most common causes of morbidity and mortality in gorillas, and it is often caused by bacteria. A thirteen-year-old captive female western lowland gorilla (Gorilla gorilla gorilla) developed hemorrhagic diarrhea. Despite the treatment, the animal died 7 days after the onset of clinical signs. The animal was submitted to a thorough pathological and microbiological evaluation. Pathologic examination revealed a severe acute hemorrhagic colitis, neutrophilic splenitis, glomerulitis, and interstitial pneumonia. Salmonella enterica serotype Infantis was isolated from a mesenteric lymph node. A diagnosis of hemorrhagic colitis associated with Salmonella enterica serotype Infantis was established. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Association of clustered regularly interspaced short palindromic repeat (CRISPR) elements with specific serotypes and virulence potential of shiga toxin-producing Escherichia coli.

PubMed

Toro, Magaly; Cao, Guojie; Ju, Wenting; Allard, Marc; Barrangou, Rodolphe; Zhao, Shaohua; Brown, Eric; Meng, Jianghong

2014-02-01

Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (P < 0.05). The relationship between the CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.

Association of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Elements with Specific Serotypes and Virulence Potential of Shiga Toxin-Producing Escherichia coli

PubMed Central

Toro, Magaly; Cao, Guojie; Ju, Wenting; Allard, Marc; Barrangou, Rodolphe; Zhao, Shaohua; Brown, Eric

2014-01-01

Shiga toxin-producing Escherichia coli (STEC) strains (n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains (n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity (P < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential (P < 0.05). The relationship between the CRISPR-cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established. PMID:24334663

Heatwaves differentially affect risk of Salmonella serotypes.

PubMed

Milazzo, Adriana; Giles, Lynne C; Zhang, Ying; Koehler, Ann P; Hiller, Janet E; Bi, Peng

2016-09-01

Given increasing frequency of heatwaves and growing public health concerns associated with foodborne disease, we examined the relationship between heatwaves and salmonellosis in Adelaide, Australia. Poisson regression analysis with Generalised Estimating Equations was used to estimate the effect of heatwaves and the impact of intensity, duration and timing on salmonellosis and specific serotypes notified from 1990 to 2012. Distributed lag non-linear models were applied to assess the non-linear and delayed effects of temperature during heatwaves on Salmonella cases. Salmonella typhimurium PT135 notifications were sensitive to the effects of heatwaves with a twofold (IRR 2.08, 95% CI 1.14-3.79) increase in cases relative to non-heatwave days. Heatwave intensity had a significant effect on daily counts of overall salmonellosis with a 34% increase in risk of infection (IRR 1.34, 95% CI 1.01-1.78) at >41 °C. The effects of temperature during heatwaves on Salmonella cases and serotypes were found at lags of up to 14 days. This study confirms heatwaves have a significant effect on Salmonella cases, and for the first time, identifies its impact on specific serotypes and phage types. These findings will contribute to the understanding of the impact of heatwaves on salmonellosis and provide insights that could mitigate their impact. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole livermore » cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.« less

Antigenic Diversity of Enteroviruses Associated with Nonpolio Acute Flaccid Paralysis, India, 2007–2009

PubMed Central

Yergolkar, Prasanna; Shankarappa, K. Subbanna

2012-01-01

Because of the broadened acute flacid paralysis (AFP) definition and enhanced surveillance, many nonpolio AFP (NP-AFP) cases have been reported in India since 2005. To determine the spectrum of nonpolio enterovirus (NPEV) serotypes associated with NP-AFP from polio-endemic and -free regions, we studied antigenic diversity of AFP-associated NPEVs. Of fecal specimens from 2,786 children with NP-AFP in 1 polio-endemic and 2 polio-free states, 823 (29.5%) were positive for NPEVs in RD cells, of which 532 (64.6%) were positive by viral protein 1 reverse transcription PCR. We identified 66 serotypes among 581 isolates, with enterovirus 71 most frequently (8.43%) detected, followed by enterovirus 13 (7.1%) and coxsackievirus B5 (5.0%). Most strains within a serotype represented new genogropups or subgenogroups. Agents for ≈35.0% and 70.0% of culture-positive and -negative cases, respectively, need to be identified. Association of human enterovirus with NP-AFP requires better assessment and understanding of health risks of NPEV infections after polio elimination. PMID:23092622

Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin.

PubMed

Skog, Johan; Mei, Ya-Fang; Wadell, Göran

2002-06-01

Most currently used adenovirus vectors are based upon adenovirus serotypes 2 and 5 (Ad2 and Ad5), which have limited efficiencies for gene transfer to human neural cells. Both serotypes bind to the known adenovirus receptor, CAR (coxsackievirus and adenovirus receptor), and have restricted cell tropism. The purpose of this study was to find vector candidates that are superior to Ad5 in infecting human neural tumours. Using flow cytometry, the vector candidates Ad4p, Ad11p and Ad17p were compared to the commonly used adenovirus vector Ad5v for their binding capacity to neural cell lines derived from glioblastoma, medulloblastoma and neuroblastoma cell lines. The production of viral structural proteins and the CAR-binding properties of the different serotypes were also assessed in these cells. Computer-based models of the fibre knobs of Ad4p and Ad17 were created based upon the crystallized fibre knob structure of adenoviruses and analysed for putative receptor-interacting regions that differed from the fibre knob of Ad5. The non CAR-binding vector candidate Ad11p showed clearly the best binding capacity to all of the neural cell lines, binding more than 90% of cells of all of the neural cell lines tested, in contrast to 20% or less for the commonly used vector Ad5v. Ad4p and Ad11p were also internalized and produced viral proteins more successfully than Ad5. Ad4p showed a low binding ability but a very efficient capacity for infection in cell culture. Ad17p virions neither bound or efficiently infected any of the neural cell lines studied.

Detection of serotype k Streptococcus mutans in Thai subjects.

PubMed

Lapirattanakul, J; Nakano, K; Nomura, R; Nemoto, H; Kojima, A; Senawongse, P; Srisatjaluk, R; Ooshima, T

2009-10-01

Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present. A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains. Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan. Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.

A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures.

PubMed

Costa, Eliane Veiga da; Campos, Renata de Mendonça; Tavares, Fernando Neto; Grégio, Cátia Regina Valério; Burlandy, Fernanda Marcicano; Silva, Edson Elias da

2012-08-01

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

A comparison of the acid-base and aciduric properties of various serotypes of the bacterium Streptococcus mutans associated with dental plaque.

PubMed

Denepitiya, L; Kleinberg, I

1984-01-01

Of the 20 strains tested, Strep. mutans Ingbritt (c), LM-7(e), QP50 -1(f) and K-1R(d/g) produced the largest decreases in pH with glucose, sucrose and starch whereas E-49(a), OMZ-61(a), FA-1(b), BHT(b), GS-5(c) and OMZ-176(d/g) produced the least. In the absence of saliva, catabolism of starch was less than catabolism of either glucose or sucrose but, with saliva present, the differences were considerably reduced. The strains varied in ability to accumulate and degrade stored polysaccharide (a means of producing a more acidic pH), but without relation to serotype. No strains showed pH-rise activity with saliva supernatant, sialin or urea but the b serotypes showed pH rise with arginine and lysylarginine . All strains showed activity from endogenous substrates. Aciduricity was determined by assessing the ability to grow on brain-heart-infusion agar adjusted to pH 5.0 to 7.0. All strains grew better at pH 7.0 than 5.0. Among the least aciduric strains were those of b serotype which being among those least able to decrease the pH with fermentable carbohydrate and the only one to raise pH with arginine and arginine peptide, suggested that this serotype might appear more often in caries-free than in caries-active plaques and in plaques showing a less acidic Stephan pH response. The acid-base and aciduric properties of the non-b serotype strains indicated that any one or more of these could be associated with caries-active plaques.

Treatment of Diabetes and Long-Term Survival After Insulin and Glucokinase Gene Therapy

PubMed Central

Callejas, David; Mann, Christopher J.; Ayuso, Eduard; Lage, Ricardo; Grifoll, Iris; Roca, Carles; Andaluz, Anna; Ruiz-de Gopegui, Rafael; Montané, Joel; Muñoz, Sergio; Ferre, Tura; Haurigot, Virginia; Zhou, Shangzhen; Ruberte, Jesús; Mingozzi, Federico; High, Katherine A.; Garcia, Felix; Bosch, Fatima

2013-01-01

Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a “glucose sensor” in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes. PMID:23378612

Clinical and microbiological characterization of serotype 6D pneumococcal infections in South Korea.

PubMed

Cheong, Hee Jin; Song, Joon Young; Choi, Min Joo; Jeon, Ji Ho; Kang, Seong Hee; Jeong, Eun Joo; Noh, Ji Yun; Kim, Woo Joo

2016-08-01

The prevalence of Serotype 6D Streptococcus pneumoniae was reported relatively high in South Korea. Since the introduction of 7-valent pneumococcal conjugate vaccine (PCV7), serotype replacement was observed. This study was designed to better clarify genetic diversity of pneumococcal serotype 6D and its clinical characteristics after introduction of PCV7 in 2000. We performed serotyping analysis with 1298 pneumococcal isolates from clinical specimens in South Korea from 2004 to 2011. Multilocus sequence typing was performed, and minimal inhibitory concentration was determined for the available serotype 6D and nontypeable (NT) pneumococcal isolates during the 2006-2007 period. The proportion of serotype 6D pneumococci increased from 0.8% (2004-2007) to 2.9% (2008-2011) of all clinical pneumococcal isolates, accounting for 14.9% of serogroup 6 pneumococci in South Korea. NT pneumococci markedly increased to 13.3% during 2006-2007 in advance of the increase in serotype 6D. Among the 26 available serotype 6D pneumococcal isolates, ST282 was predominant (23 isolates, 88.5%). The STs of NT pneumococci (26 isolates) were diverse, but clonal complex 271 was the dominant clone. The oral penicillin non-susceptibility rate was 92.3% (24 among 26 isolates) for both serotype 6D and NT pneumococci. The ceftriaxone non-susceptibility rates of serotype 6D and NT pneumococci were 7.7% and 3.8%, respectively. ST228(6D) strain expanded, particularly among old adults with comorbidities in South Korea. Both antibiotic and PCV7 pressure might have contributed to the selective increase of NT and serotype 6D pneumococci. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction.

PubMed

Furtak, Vyacheslav; Roivainen, Merja; Mirochnichenko, Olga; Zagorodnyaya, Tatiana; Laassri, Majid; Zaidi, Sohail Z; Rehman, Lubna; Alam, Muhammad M; Chizhikov, Vladimir; Chumakov, Konstantin

2016-04-14

An approach is proposed for environmental surveillance of poliovirus by concentrating sewage samples with tangential flow filtration (TFF) followed by deep sequencing of viral RNA. Subsequent to testing the method with samples from Finland, samples from Pakistan, a country endemic for poliovirus, were investigated. Genomic sequencing was either performed directly, for unbiased identification of viruses regardless of their ability to grow in cell cultures, or after virus enrichment by cell culture or immunoprecipitation. Bioinformatics enabled separation and determination of individual consensus sequences. Overall, deep sequencing of the entire viral population identified polioviruses, non-polio enteroviruses, and other viruses. In Pakistani sewage samples, adeno-associated virus, unable to replicate autonomously in cell cultures, was the most abundant human virus. The presence of recombinants of wild polioviruses of serotype 1 (WPV1) was also inferred, whereby currently circulating WPV1 of south-Asian (SOAS) lineage comprised two sub-lineages depending on their non-capsid region origin. Complete genome analyses additionally identified point mutants and intertypic recombinants between attenuated Sabin strains in the Pakistani samples, and in one Finnish sample. The approach could allow rapid environmental surveillance of viruses causing human infections. It creates a permanent digital repository of the entire virome potentially useful for retrospective screening of future discovered viruses.

Release of Bioactive Adeno-Associated Virus from Fibrin Scaffolds: Effects of Fibrin Glue Concentrations

PubMed Central

Lee, Hannah H.; Haleem, Amgad M.; Yao, Veronica; Li, Juan; Xiao, Xiao

2011-01-01

Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. However, the effect of different fibrinogen concentrations on FG scaffold delivery of bioactive adeno-associated viruses (AAVs) has not been established. This study was performed to test the hypothesis that FG concentration alters AAV release profiles, which affect AAV bioavailability. Gene transfer efficiency of AAV-GFP released from FG was measured using HEK-293 cells. Bioactivity of AAV transforming growth factor-beta1 (TGF-β1) released from FG was assessed using the mink lung cell assay, and by measuring induction of cartilage-specific gene expression in human mesenchymal stem cells (hMSCs). Nondiluted FG had longer clotting times, smaller pore sizes, thicker fibers, and slower dissolution rate, resulting in reduced release of AAV. AAV release and gene transfer efficiency was higher with 25% and 50% FG than with the 75% and 100% FG. AAV-TGF-β1 released from dilute-FG transduced hMSCs, resulting in higher concentrations of bioactive TGF-β1 and greater upregulation of cartilage-specific gene expression compared with hMSC from undiluted FG. This study, showing improved release, transduction efficiency, and chondrogenic effect on hMSC of bioactive AAV-TGF-β1 released from diluted FG, provides information important to optimization of this clinically available scaffold for therapeutic gene delivery, both in cartilage regeneration and for other tissue engineering applications. PMID:21449684

Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

PubMed

van Lieshout, Laura P; Soule, Geoff; Sorensen, Debra; Frost, Kathy L; He, Shihua; Tierney, Kevin; Safronetz, David; Booth, Stephanie A; Kobinger, Gary P; Qiu, Xiangguo; Wootton, Sarah K

2018-03-05

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

Around the World in 1,475 Salmonella Geo-serotypes

PubMed Central

Le Hello, Simon; de Jong, Birgitta; Rolfhamre, Per; Faensen, Daniel; Weill, François-Xavier; Giesecke, Johan

2016-01-01

It’s easy to remember Salmonella serotypes names, isn’t it? Surely, this is because the naming system of Salmonella serotypes is by far the most scientist friendly. Traditionally, most Salmonella serotypes have been named after geographic locations. We decided to explore the geographic locations to which Salmonella serotypes refer and describe some unexpected twists in the naming scheme. We found that 93% (n = 1,475) of the 1,585 serotypes could be categorized as geo-serotypes; that is, the name refers to a geographic location. The 3 countries with the most geo-serotypes are Germany, the United Kingdom, and the United States. Other serotype names refer to the name of a person, animal, tribe, or food item or are a composite of symptoms and host. The Salmonella serotypes naming scheme has had a valuable effect on public health microbiology, and in the current era of fast development of whole-genome sequencing, it should remain a reference.

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

PubMed Central

Nivarthi, Usha K.; Kose, Nurgun; Sapparapu, Gopal; Widman, Douglas; Gallichotte, Emily; Pfaff, Jennifer M.; Doranz, Benjamin J.; Weiskopf, Daniela; Sette, Alessandro; Durbin, Anna P.; Whitehead, Steve S.; Baric, Ralph

2016-01-01

responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines. PMID:28031369

Preclinical evaluation of radiation and systemic, RGD-targeted, adeno-associated virus phage-TNF gene therapy in a mouse model of spontaneously metastatic melanoma.

PubMed

Quinn, T J; Healy, N; Sara, A; Maggi, E; Claros, C S; Kabarriti, R; Scandiuzzi, L; Liu, L; Gorecka, J; Adem, A; Basu, I; Yuan, Z; Guha, C

2017-01-01

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction.

PubMed

Shao, Wenwei; Earley, Lauriel F; Chai, Zheng; Chen, Xiaojing; Sun, Junjiang; He, Ting; Deng, Meng; Hirsch, Matthew L; Ting, Jenny; Samulski, R Jude; Li, Chengwen

2018-06-21

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6-10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Antimicrobial Properties of Biofunctionalized Silver Nanoparticles on Clinical Isolates of Streptococcus mutans and Its Serotypes

PubMed Central

Martínez-Robles, Ángel Manuel; Loyola-Rodríguez, Juan Pablo; Zavala-Alonso, Norma Verónica; Martinez-Martinez, Rita Elizabeth; Ruiz, Facundo; Lara-Castro, René Homero; Donohué-Cornejo, Alejandro; Reyes-López, Simón Yobanny; Espinosa-Cristóbal, León Francisco

2016-01-01

(1) Background: Streptococcus mutans (S. mutans) is the principal pathogen involved in the formation of dental caries. Other systemic diseases have also been associated with specific S. mutans serotypes (c, e, f, and k). Silver nanoparticles (SNP) have been demonstrated to have good antibacterial effects against S. mutans; therefore, limited studies have evaluated the antimicrobial activity of biofunctionalized SNP on S. mutans serotypes. The purpose of this work was to prepare and characterize coated SNP using two different organic components and to evaluate the antimicrobial activity of SNP in clinical isolates of S. mutans strains and serotypes; (2) Methods: SNP with bovine serum albumin (BSA) or chitosan (CS) coatings were prepared and the physical, chemical and microbiological properties of SNP were evaluated; (3) Results: Both types of coated SNP showed antimicrobial activity against S. mutans bacteria and serotypes. Better inhibition was associated with smaller particles and BSA coatings; however, no significant differences were found between the different serotypes, indicating a similar sensitivity to the coated SNP; (4) Conclusion: This study concludes that BSA and CS coated SNP had good antimicrobial activity against S. mutans strains and the four serotypes, and this study suggest the widespread use of SNP as an antimicrobial agent for the inhibition of S. mutans bacteria. PMID:28335264

Antimicrobial Properties of Biofunctionalized Silver Nanoparticles on Clinical Isolates of Streptococcus mutans and Its Serotypes.

PubMed

Martínez-Robles, Ángel Manuel; Loyola-Rodríguez, Juan Pablo; Zavala-Alonso, Norma Verónica; Martinez-Martinez, Rita Elizabeth; Ruiz, Facundo; Lara-Castro, René Homero; Donohué-Cornejo, Alejandro; Reyes-López, Simón Yobanny; Espinosa-Cristóbal, León Francisco

2016-07-22

(1) Background: Streptococcus mutans ( S. mutans ) is the principal pathogen involved in the formation of dental caries. Other systemic diseases have also been associated with specific S. mutans serotypes ( c , e , f , and k ). Silver nanoparticles (SNP) have been demonstrated to have good antibacterial effects against S. mutans ; therefore, limited studies have evaluated the antimicrobial activity of biofunctionalized SNP on S. mutans serotypes. The purpose of this work was to prepare and characterize coated SNP using two different organic components and to evaluate the antimicrobial activity of SNP in clinical isolates of S. mutans strains and serotypes; (2) Methods: SNP with bovine serum albumin (BSA) or chitosan (CS) coatings were prepared and the physical, chemical and microbiological properties of SNP were evaluated; (3) Results: Both types of coated SNP showed antimicrobial activity against S. mutans bacteria and serotypes. Better inhibition was associated with smaller particles and BSA coatings; however, no significant differences were found between the different serotypes, indicating a similar sensitivity to the coated SNP; (4) Conclusion: This study concludes that BSA and CS coated SNP had good antimicrobial activity against S. mutans strains and the four serotypes, and this study suggest the widespread use of SNP as an antimicrobial agent for the inhibition of S. mutans bacteria.

Assessing risk profiles for Salmonella serotypes in breeding pig operations in Portugal using a Bayesian hierarchical model.

PubMed

Correia-Gomes, Carla; Economou, Theodoros; Mendonça, Denisa; Vieira-Pinto, Madalena; Niza-Ribeiro, João

2012-11-21

The EU Regulation No 2160/2003 imposes a reduction in the prevalence of Salmonella in pigs. The efficiency of control programmes for Salmonella in pigs, reported among the EU Member States, varies and definitive eradication seems very difficult. Control measures currently recommended for Salmonella are not serotype-specific. Is it possible that the risk factors for different Salmonella serotypes are different? The aim of this study was to investigate potential risk factors for two groups of Salmonella sp serotypes using pen faecal samples from breeding pig holdings representative of the Portuguese pig sector. The data used come from the Baseline Survey for the Prevalence of Salmonella in breeding pigs in Portugal. A total of 1670 pen faecal samples from 167 herds were tested, and 170 samples were positive for Salmonella. The presence of Salmonella in each sample (outcome variable) was classified in three categories: i) no Salmonella, ii) Salmonella Typhimurium or S. Typhimurium-like strains with the antigenic formula: 1,4,5,12:i:-, , and iii) other serotypes. Along with the sample collection, a questionnaire concerning herd management and potential risk factors was utilised. The data have a "natural" hierarchical structure so a categorical multilevel analysis of the dataset was carried out using a Bayesian hierarchical model. The model was estimated using Markov Chain Monte Carlo methods, implemented in the software WinBUGS. The significant associations found (when compared to category "no Salmonella"), for category "serotype Typhimurium or S. Typhimurium-like strains with the antigenic formula: 1,4,5,12:i:-" were: age of breeding sows, size of the herd, number of pigs/pen and source of semen. For the category "other serotypes" the significant associations found were: control of rodents, region of the country, source of semen, breeding sector room and source of feed. The risk factors significantly associated with Salmonella shedding from the category "serotype

Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trempe, J.P.; Carter, B.J.

1988-01-01

The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/submore » 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.« less

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

PubMed

Verdezoto, J; Breard, E; Viarouge, C; Quenault, H; Lucas, P; Sailleau, C; Zientara, S; Augot, D; Zapata, S

2018-02-01

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador. © 2017 Blackwell Verlag GmbH.

Immune Response and Viral Persistence in Indian Buffaloes (Bubalus bubalis) Infected with Foot-and-Mouth Disease Virus Serotype Asia 1 ▿

PubMed Central

Maddur, Mohan S.; Kishore, Subodh; Gopalakrishna, S.; Singh, Nem; Suryanarayana, V. V.; Gajendragad, Mukund R.

2009-01-01

Despite their potential role in the spread of foot-and-mouth disease (FMD), the immune response and viral persistence in FMD virus (FMDV)-infected Indian buffaloes (Bubalus bubalis) have been unexplored. We found similar kinetics of neutralizing antibody responses in the sera and secretory fluids of buffaloes following experimental FMDV Asia 1 infection, but the lymphocyte-proliferative response in infected buffaloes was of low magnitude. Despite inducing a significant systemic and secretory immune response, viral persistence seems to be a common outcome in buffaloes following FMDV Asia 1 infection, which is associated with a weak cellular immune response. PMID:19828770

RNA polymerase activity is associated with viral particles isolated from Leishmania braziliensis subsp. guyanensis.

PubMed Central

Widmer, G; Keenan, M C; Patterson, J L

1990-01-01

Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found. Images PMID:2370680

Disinfectant susceptibility of different Salmonella serotypes isolated from chicken and egg production chains.

PubMed

Long, M; Lai, H; Deng, W; Zhou, K; Li, B; Liu, S; Fan, L; Wang, H; Zou, L

2016-09-01

The study aimed to serotype the Salmonella isolates recovered from chicken and egg production chains, and to investigate the disinfectant resistance phenotypes and genotypes of these isolates. The Salmonella isolates were serotyped, and the minimal inhibitory concentrations (MICs) of disinfectants were determined. Results showed that the Salmonella isolates recovered from both chains were diverse, and the serotypes in each part of the production chain and between the two production chains were significantly different. In the chicken production chain, 19 different serotypes were recovered, while only five serotypes were found in the egg production chain. The isolates showed a high susceptibility to didecyldimethylammonium bromide (DDAB) but a low susceptibility to benzalkonium chloride (BC), benzalkonium bromide (BAB) and chlorhexidine (CHX). Salmonella Enteritidis and Salmonella Typhimurium were more resistant to BC and BAB. The qacEΔ1 and qacF resistance genes were detected in 26·7 and 7·7% of the isolates respectively. The qacEΔ1 gene was frequently found in Salmonella Derby and Salm. Enteritidis (P < 0·05). Our findings indicated that Salmonella was commonly present in both chains, and could serve as a critical vector in spreading disinfectant resistance associated with different serotypes. This study first demonstrated disinfectant resistance phenotypes and genotypes of serotyped Salmonella. The study highlights the need for monitoring the disinfectant resistance varied in different Salmonella serotypes. © 2016 The Society for Applied Microbiology.

Longitudinal survey of the distribution of various serotypes of Streptococcus mutans in infants.

PubMed

Masuda, N; Tsutsumi, N; Sobue, S; Hamada, S

1979-10-01

The establishment of various serotypes of Streptococcus mutans was studied serologically in plaque samples collected from label surfaces of upper primary incisors of 22 infants (starting age, 5 to 13 months) over a period fo 30 months. Clinical examinations were also performed. No clear-cut association between the initiation of dental caries and previous detection of S. mutans was noted. However, all 12 of the infants with caries had S. mutans isolated at some time during the course of this study. The most common serotype isolated at the initial establishment of S. mutans on the tooth surfaces was serotype c, whereas types d, e, and g became established in a few cases. During the test period, changes in the distribution of serotypes of S. mutans were observed in some cases. The initiation of carious lesions could be found in a few cases even when S. mutans comprised about 1% or less of the total streptococcal count of the specimen from the tooth surfaces. Serotype d/g strains tended to develop carious lesions on smooth surfaces, although serotype c was isolated from almost all individuals who developed caries.

Streptococcus agalactiae serotype Ib as an agent of meningitis in two adult nonpregnant women.

PubMed

Martins, E R; Florindo, C; Martins, F; Aldir, I; Borrego, M J; Brum, L; Ramirez, M; Melo-Cristino, J

2007-11-01

Two temporally and geographically clustered cases of meningitis caused by Streptococcus agalactiae expressing the infrequent Ib serotype are reported. Characterization by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the isolates were identical and represented the widely distributed ST10/ST8 lineage associated with serotype Ib.

Determination of evolutionary relationships of outbreak-associated Listeria monocytogenes strains of serotypes 1/2a and 1/2b by whole-genome sequencing

USDA-ARS?s Scientific Manuscript database

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or “epidemic clones” that have been previously associated with outb...

Serotype- and virulence-associated gene profile of Streptococcus suis isolates from pig carcasses in Chiang Mai Province, Northern Thailand

PubMed Central

WONGSAWAN, Kanruethai; GOTTSCHALK, Marcelo; THARAVICHITKUL, Prasit

2014-01-01

In this present study, the serotype of 40 Streptococcus suis isolates from submaxillary glands of pig carcasses sold in wet markets in Chiang Mai Province, northern Thailand, was investigated. Eleven serotypes, including types 2, 3, 4, 5, 7, 8, 9, 17, 21, 22 and 31, were found in the isolates by a Multiplex PCR combined with serum agglutination. Of the eleven serotypes present, type 3 was the most prevalent, while types 2, 4, 5 and 21 were of primary interest due to their human isolate serotype. The mrp+/epf − /sly − genotype was found to be the most prevalent genotype. This study indicates the importance of effective control of human S. suis infection due to raw pork or pig carcass handling in northern Thailand. PMID:25367105

Serotype- and virulence-associated gene profile of Streptococcus suis isolates from pig carcasses in Chiang Mai Province, Northern Thailand.

PubMed

Wongsawan, Kanruethai; Gottschalk, Marcelo; Tharavichitkul, Prasit

2015-02-01

In this present study, the serotype of 40 Streptococcus suis isolates from submaxillary glands of pig carcasses sold in wet markets in Chiang Mai Province, northern Thailand, was investigated. Eleven serotypes, including types 2, 3, 4, 5, 7, 8, 9, 17, 21, 22 and 31, were found in the isolates by a Multiplex PCR combined with serum agglutination. Of the eleven serotypes present, type 3 was the most prevalent, while types 2, 4, 5 and 21 were of primary interest due to their human isolate serotype. The mrp+/epf - /sly - genotype was found to be the most prevalent genotype. This study indicates the importance of effective control of human S. suis infection due to raw pork or pig carcass handling in northern Thailand.

Stochastical modeling for Viral Disease: Statistical Mechanics and Network Theory

NASA Astrophysics Data System (ADS)

Zhou, Hao; Deem, Michael

2007-04-01

Theoretical methods of statistical mechanics are developed and applied to study the immunological response against viral disease, such as dengue. We use this theory to show how the immune response to four different dengue serotypes may be sculpted. It is the ability of avian influenza, to change and to mix, that has given rise to the fear of a new human flu pandemic. Here we propose to utilize a scale free network based stochastic model to investigate the mitigation strategies and analyze the risk.

Comprehensive analysis of Salmonella sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes.

PubMed

Lauri, Andrea; Castiglioni, Bianca; Mariani, Paola

2011-07-01

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction-Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.

Novel Ganglioside-mediated Entry of Botulinum Neurotoxin Serotype D into Neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kroken, Abby R.; Karalewitz, Andrew P.-A.; Fu, Zhuji

2012-02-07

Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique. In this study BoNT/D is shown to have a unique association with ganglioside relative to the other BoNT serotypes, utilizing amore » ganglioside binding loop (GBL, residues Tyr-1235-Ala-1245) within the receptor binding domain of BoNT/D (HCR/D) via b-series gangliosides, including GT1b, GD1b, and GD2. HCR/D bound gangliosides and entered neurons dependent upon the aromatic ring of Phe-1240 within the GBL. This is the first BoNT-ganglioside interaction that is mediated by a phenylalanine. In contrast, Trp-1238, located near the N terminus of the ganglioside binding loop, was mostly solvent-inaccessible and appeared to contribute to maintaining the loop structure. BoNT/D entry and intoxication were enhanced by membrane depolarization via synaptic vesicle cycling, where HCR/D colocalized with synaptophysin, a synaptic vesicle marker, but immunoprecipitation experiments did not detect direct association with synaptic vesicle protein 2. Thus, BoNT/D utilizes unique associations with gangliosides and synaptic vesicles to enter neurons, which may facilitate new neurotoxin therapies.« less

Chlamydia trachomatis serotype A infections in the Amazon region of Brazil: prevalence, entry and dissemination.

PubMed

Ishak, Marluísa de Oliveira Guimarães; Costa, Maurimélia Mesquita; Almeida, Núbia Caroline Costa de; Santiago, Angélica Menezes; Brito, William Botelho de; Vallinoto, Antonio Carlos Rosário; Azevedo, Vânia Nakauth; Ishak, Ricardo

2015-01-01

Chlamydia infection is associated with debilitating human diseases including trachoma, pneumonia, coronary heart disease and urogenital diseases. Serotypes of C. trachomatis show a fair correlation with the group of diseases they cause, and their distribution follows a well-described geographic pattern. Serotype A, a trachoma-associated strain, is known for its limited dissemination in the Middle East and Northern Africa. However, knowledge on the spread of bacteria from the genus Chlamydia as well as the distribution of serotypes in Brazil is quite limited. Blood samples of 1,710 individuals from ten human population groups in the Amazon region of Brazil were examined for antibodies to Chlamydia using indirect immunofluorescence and microimmunofluorescence assays. The prevalence of antibodies to Chlamydia ranged from 23.9% (Wayana-Apalai) to 90.7% (Awa-Guaja) with a mean prevalence of 50.2%. Seroreactivity was detected to C. pneumoniae and to all serotypes of C. trachomatis tested; furthermore, we report clear evidence of the as-yet-undescribed occurrence of serotype A of C. trachomatis. Specific seroreactivity not only accounts for the large extent of dissemination of C. trachomatis in the Amazon region of Brazil but also shows an expanded area of occurrence of serotype A outside the epidemiological settings previously described. Furthermore, these data suggest possible routes of Chlamydia introduction into the Amazon region from the massive human migration that occurred during the 1,700s.

Nonalcoholic fatty liver disease and hepatic cirrhosis: Comparison with viral hepatitis-associated steatosis.

PubMed

Haga, Yuki; Kanda, Tatsuo; Sasaki, Reina; Nakamura, Masato; Nakamoto, Shingo; Yokosuka, Osamu

2015-12-14

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) is globally increasing and has become a world-wide health problem. Chronic infection with hepatitis B virus or hepatitis C virus (HCV) is associated with hepatic steatosis. Viral hepatitis-associated hepatic steatosis is often caused by metabolic syndrome including obesity, type 2 diabetes mellitus and/or dyslipidemia. It has been reported that HCV genotype 3 exerts direct metabolic effects that lead to hepatic steatosis. In this review, the differences between NAFLD/NASH and viral hepatitis-associated steatosis are discussed.

Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1

PubMed Central

Deng, Haoyu; Fung, Gabriel; Shi, Junyan; Xu, Suowen; Wang, Chen; Yin, Meimei; Hou, Jun; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

2015-01-01

Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2-associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus-encoded protease 2Apro, independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus-induced cleavage of GAB1 is beneficial to viral growth as the N-terminal proteolytic product of GAB1 (GAB1-N1–174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1-N1–174 fragment that enhances viral infectivity, at least in part, via activation of the ERK pathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication.—Deng, H., Fung, G., Shi, J., Xu, S., Wang, C., Yin, M., Hou, J., Zhang, J., Jin, Z.-G., Luo, H. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1. PMID:26183772

Serotype changes in adult invasive pneumococcal infections in Portugal did not reduce the high fraction of potentially vaccine preventable infections.

PubMed

Horácio, Andreia N; Diamantino-Miranda, Jorge; Aguiar, Sandra I; Ramirez, Mário; Melo-Cristino, José

2012-01-05

We determined the serotype and antimicrobial susceptibility of 1100 isolates responsible for adult invasive pneumococcal infections (IPD) in Portugal between 2006 and 2008. Serotypes 3 (13%), 1 (12%), 7F (11%), 19A (10%) and 14 (7%) were the most frequent causes of IPD and the two later serotypes accounted for the majority of erythromycin and penicillin nonsusceptible isolates. Serotype 1 was associated with younger adults whereas serotype 3 was associated with older adults. Despite the availability of the 23-valent polysaccharide vaccine (PPV23) in Portugal since 1996, the proportion of PPV23 preventable IPD remained stable and above 80%. Comparing with previous data from Portugal, we showed a continued decline of the serotypes included in the 7-valent conjugate vaccine (PCV7) in adult IPD and a rise of serotypes included in the 13-valent conjugate vaccine, increasing its potential coverage of adult IPD to 70% in 2008. Penicillin non-susceptibility remained stable (17%) whereas erythromycin resistance (18%) has continued to rise in the post-PCV7 years. Copyright © 2011 Elsevier Ltd. All rights reserved.

Rapid molecular pathotyping of major salmonella enterica serotypes based on single-nucleotide polymorphisms (SNPs) in the adenylate cyclase (cyaA) gene

USDA-ARS?s Scientific Manuscript database

Introduction: Salmonella enterica subsp. enterica serotype Enteriditis (S. Enteriditis) is the leading cause of salmonellosis worldwide, including the USA. Many S. enterica serotypes known to cause foodborne disease are associated with broiler meat contamination. While some serotypes are specific...

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

PubMed

Hiramoto, Takafumi; Li, Li B; Funk, Sarah E; Hirata, Roli K; Russell, David W

2018-05-02

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4 + and CD8 + T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies

PubMed Central

Yu, Jigui; Lin, Jisheng; Benjamin, William H.; Waites, Ken B.; Lee, Che-hung; Nahm, Moon H.

2005-01-01

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy. PMID:15634965

Pediatric Invasive Pneumococcal Disease in Guatemala City: Importance of Serotype 2.

PubMed

Gaensbauer, James T; Asturias, Edwin J; Soto, Monica; Holt, Elizabeth; Olson, Daniel; Halsey, Neal A

2016-05-01

To inform estimations of the potential impact of recently introduced pneumococcal conjugate vaccine (PCV), we report results of 11 years of pre-PCV surveillance for invasive pneumococcal disease (IPD) among children in Guatemala City. Cases of IPD in children younger than 5 years were identified by active surveillance at 3 referral hospitals in Guatemala City from October 1996 through 2007. Clinical and demographic data were obtained, and isolates of Streptococcus pneumoniae from normally sterile sites were serotyped using latex agglutination and confirmed by Quellung reaction. Four hundred fifty-two cases of IPD were identified with a case fatality rate of 21%. Meningitis was the most common cause of death (77% of all deaths) and occurred more often in infancy (median age 5 months) than other clinical syndromes. Of the 137 isolates serotyped, type 1 (26 cases, 17%), type 2 (25 cases, 16%) and type 5 (18 cases, 12%) were the most common. Serotype 2 was associated with a higher case fatality rate (28%), higher rate of meningitis (68%) and occurred in younger infants (median age, 3.5 months) than other common serotypes. Recently introduced PCV13 includes 73% of observed serotypes in the study. Infants with IPD presented at a young age. Serotype 2, rarely reported as a significant cause of IPD and not included in available PCVs, was a common cause of disease in this population. PCV13 introduction in Guatemala, begun in 2013, may not have as great an impact in disease reduction as has been observed in other countries.

Serotypes in Saccharomyces telluris: Their relation to source of isolation

USGS Publications Warehouse

Hasenclever, H.F.; Kocan, R.M.

1973-01-01

Three serotypes have been characterized with three reference strains of Saccharomyces telluris and designated as A, B, and C. One reference strain of Torpulopsis bovina, the imperfect form of S. telluris, belonged to serotype B. Strains of S. telluris isolated from four columbid species were serotyped. All 98 strains of this yeast isolated from Columba livia belonged to serotype B. Three other columbid species, C. leucocephala, C. fasciata, and Zenaidura macroura harbored strains of serotype C only. Serotype A was not isolated from any of the avian species.

Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption.

PubMed

Johansen, Anne Katrine; Molenaar, Bas; Versteeg, Danielle; Leitoguinho, Ana Rita; Demkes, Charlotte; Spanjaard, Bastiaan; de Ruiter, Hesther; Akbari Moqadam, Farhad; Kooijman, Lieneke; Zentilin, Lorena; Giacca, Mauro; van Rooij, Eva

2017-10-27

CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized. To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice. We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6 , Sav1 , and Tbx20 , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1 , which increased the editing efficiency. Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo. © 2017 American Heart Association, Inc.

Antimicrobial resistance, serotypes, and virulence factors of Streptococcus suis isolates from diseased pigs.

PubMed

Li, Lu-Lu; Liao, Xiao-Ping; Sun, Jian; Yang, Yu-Rong; Liu, Bao-Tao; Yang, Shou-Shen; Zhao, Dong-Hao; Liu, Ya-Hong

2012-07-01

Streptococcus suis isolates from diseased pigs were examined for susceptibility to nine antimicrobials, possession of virulence-associated factors (VFs), and distribution of serotypes. The association between antimicrobial resistance (AMR) and serotypes as well as VFs was subsequently assessed. Among the isolates investigated, serotype 2 (66.04%) was mostly prevalent, followed by serotypes 1 (23.27%), 9 (1.26%), and 7 (0.63%), whereas 14 isolates were untypable by the polymerase chain reaction typing method used. Analysis with pulsed-field gel electrophoresis revealed the isolates had diverse DNA macrorestriction patterns. The frequency of antimicrobial resistance among the S. suis isolates was higher than that reported from other countries. It is notable that multiple antimicrobial resistance (three or more antimicrobials) was observed with 98.73% of the S. suis isolates, and the dominant resistance phenotype was erythromycin-tilmicosin-clindamycin-chloramphenicol-levofloxacin-ceftiofur-kanamycin-tetracycline-penicillin (35.85%). The most prevalent VFs were those encoded by muramidase-released protein (61.64%), followed by suilysin (56.60%) and extracellular factor (46.54%). Presence of VFs and the possession of certain AMR phenotypes were significantly associated as determined by statistical analysis. Together, these findings indicate that the clinical S. suis isolates obtained from diseased pigs in China are genetically diverse, are resistant to multiple antibiotics of clinical importance, and carry known virulence factors.

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

PubMed Central

Leung, Marcus H.; Bryson, Kevin; Freystatter, Kathrin; Pichon, Bruno; Edwards, Giles; Gillespie, Stephen H.

2012-01-01

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future. PMID:22553238

Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

PubMed

Karalewitz, Andrew P-A; Fu, Zhuji; Baldwin, Michael R; Kim, Jung-Ja P; Barbieri, Joseph T

2012-11-23

How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear. BoNT/C utilizes dual gangliosides as host cell receptors. BoNT/C accesses gangliosides on the plasma membrane. Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.

Associations between national viral hepatitis policies/programmes and country-level socioeconomic factors: a sub-analysis of data from the 2013 WHO viral hepatitis policy report.

PubMed

Lazarus, Jeffrey V; Sperle, Ida; Safreed-Harmon, Kelly; Gore, Charles; Cebolla, Beatriz; Spina, Alexander

2017-07-26

As more countries worldwide develop national viral hepatitis strategies, it is important to ask whether context-specific factors affect their decision-making. This study aimed to determine whether country-level socioeconomic factors are associated with viral hepatitis programmes and policy responses across WHO Member States (MS). WHO MS focal points completed a questionnaire on national viral hepatitis policies. This secondary analysis of data reported in the 2013 Global Policy Report on the Prevention and Control of Viral Hepatitis in WHO Member States used logistic regression to examine associations between four survey questions and four socioeconomic factors: country income level, Human Development Index (HDI), health expenditure and physician density. This analysis included 119 MS. MS were more likely to have routine viral hepatitis surveillance and to have a national strategy and/or policy/guidelines for preventing infection in healthcare settings if they were in the higher binary categories for income level, HDI, health expenditure and physician density. In multivariable analyses, the only significant finding was a positive association between having routine surveillance and being in the higher binary HDI category (adjusted odds ratio 26; 95% confidence interval 2.0-340). Countries with differing socioeconomic status indicators did not appear to differ greatly regarding the existence of key national policies and programmes. A more nuanced understanding of the multifaceted interactions of socioeconomic factors, health policy, service delivery and health outcomes is needed to support country-level efforts to eliminate viral hepatitis.

Dominance of serotype Ia among group B Streptococci causing invasive infections in nonpregnant adults in Portugal.

PubMed

Martins, E R; Melo-Cristino, J; Ramirez, M

2012-04-01

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

Antigen analyses of serotypes of streptococcus mutans using a monoclonal antibody elaborated against serotype g polysaccharide antigen.

PubMed

Okahashi, N; Nishida, Y; Futakami, K; Hamada, S

1985-04-01

A hybridoma (F4B) which produced a monoclonal antibody (mAb) specific for serotype g carbohydrate antigen (RRg) of Streptococcus mutans 6715 was obtained. The F4B mAb cross-reacted with purified carbohydrate antigens of serotype d (RRd) and serotype h (TCAh). In immunodiffusion tests, F4B mAb produced a stable precipitin band with RRg, while the band developed between the mAb and RRd/TCAh in the cold disappeared when incubated at room temperature. The immunoprecipitin reaction between F4B mAb and RRg was strongly inhibited upon addition of lactose.

Serotypic characterization of rotaviruses derived from asymptomatic human neonatal infections.

PubMed Central

Hoshino, Y; Wyatt, R G; Flores, J; Midthun, K; Kapikian, A Z

1985-01-01

Nineteen rotavirus strains derived from asymptomatic neonates (seven from England, five from Australia, two from Venezuela, and five from Sweden) were successfully cultivated in primary African green monkey kidney cell cultures, serotyped by plaque reduction neutralization tests, subgrouped by indirect enzyme-linked immunosorbent assay, and electropherotyped by polyacrylamide gel electrophoresis. All 19 strains were shown to fall into one of the four known human serotypes; serotype 1 (all Venezuelan strains), serotype 2 (all Swedish strains), serotype 3 (all Australian strains), or serotype 4 (all English strains). Hyperimmune guinea pig serum raised against the Venezuelan strain (M37) neutralized not only serotype 1 (strain Wa) but also serotype 4 (strain St. Thomas no. 3) viruses to a similar degree. The English, Australian, and Venezuelan isolates were found to belong to subgroup 2, and the Swedish strains were subgroup 1 viruses. The potential importance of these rotaviruses obtained from neonates as possible vaccine candidates is discussed. Images PMID:2984247

Serotype-specific pneumococcal antibody concentrations in children treated for acute leukaemia.

PubMed

Patel, Soonie R; Bate, Jessica; Borrow, Ray; Heath, Paul T

2012-01-01

Children treated for acute leukaemia are at increased risk of infection with Streptococcus pneumoniae. The basis for this may include low levels of pneumococcal antibody but this has not been well studied. The authors measured serotype-specific pneumococcal IgG antibody concentrations in children treated for acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) ≥6 months after completion of standard-dose chemotherapy. Pneumococcal serotype-specific IgG antibody concentrations were low. None of the subjects had protective concentrations against all the heptavalent-pneumococcal conjugate vaccine serotypes. There was no significant difference in antibody concentrations between subjects with ALL and AML (p≥0.05). Children treated for ALL and AML generally have non-protective antibody concentrations against S pneumoniae. There is significant morbidity associated with pneumococcal disease in this patient group and strategies for vaccination are required.

Viral vector-based reversible neuronal inactivation and behavioral manipulation in the macaque monkey

PubMed Central

Nielsen, Kristina J.; Callaway, Edward M.; Krauzlis, Richard J.

2012-01-01

Viral vectors are promising tools for the dissection of neural circuits. In principle, they can manipulate neurons at a level of specificity not otherwise achievable. While many studies have used viral vector-based approaches in the rodent brain, only a few have employed this technique in the non-human primate, despite the importance of this animal model for neuroscience research. Here, we report evidence that a viral vector-based approach can be used to manipulate a monkey's behavior in a task. For this purpose, we used the allatostatin receptor/allatostatin (AlstR/AL) system, which has previously been shown to allow inactivation of neurons in vivo. The AlstR was expressed in neurons in monkey V1 by injection of an adeno-associated virus 1 (AAV1) vector. Two monkeys were trained in a detection task, in which they had to make a saccade to a faint peripheral target. Injection of AL caused a retinotopic deficit in the detection task in one monkey. Specifically, the monkey showed marked impairment for detection targets placed at the visual field location represented at the virus injection site, but not for targets shown elsewhere. We confirmed that these deficits indeed were due to the interaction of AlstR and AL by injecting saline, or AL at a V1 location without AlstR expression. Post-mortem histology confirmed AlstR expression in this monkey. We failed to replicate the behavioral results in a second monkey, as AL injection did not impair the second monkey's performance in the detection task. However, post-mortem histology revealed a very low level of AlstR expression in this monkey. Our results demonstrate that viral vector-based approaches can produce effects strong enough to influence a monkey's performance in a behavioral task, supporting the further development of this approach for studying how neuronal circuits control complex behaviors in non-human primates. PMID:22723770

Oxybuprocaine induces a false-positive response in immunochromatographic SAS Adeno Test.

PubMed

Hoshino, Takeshi; Takanashi, Taiji; Okada, Morio; Uchida, Sunao

2002-04-01

To investigate whether a solution of oxybuprocaine hydrochloride, 0.4%, results in a false-positive response in an immunochromatographic SAS Adeno Test. Experimental study. Physiologic saline and 2% lidocaine. Each chemical (100 microl) was diluted in a transport medium. Five drops (200 microl) of the resultant solution were dispensed into the round sample well of a test device. Fifteen samples were tested in each group. Ten minutes after the start of the test, a colored line in the "specimen" portion of the test membrane was visually read as positive or negative by a masked technician. No positive reaction was observed in the control groups (physiologic saline and lidocaine). A false-positive reaction was observed in six samples (33.3%) in the oxybuprocaine group. The positive rate was significantly higher in the oxybuprocaine group compared with those in control groups (P = 0.0062, Fisher's extract probability test). Oxybuprocaine may induce a false-positive reaction in an immunochromatographic SAS Adeno Test. We recommend the use of lidocaine, instead of oxybuprocaine, for local anesthesia in taking eye swabs from patients with suspected adenovirus infection.

Individual and community factors associated with geographic clusters of poor HIV care retention and poor viral suppression

PubMed Central

Eberhart, Michael G.; Yehia, Baligh R.; Hillier, Amy; Voytek, Chelsea D.; Fiore, Danielle J.; Blank, Michael; Frank, Ian; Metzger, David S.; Brady, Kathleen A.

2015-01-01

Background Previous analyses identified specific geographic areas in Philadelphia (hotspots) associated with negative outcomes along the HIV care continuum. We examined individual and community factors associated with residing in these hotspots. Methods Retrospective cohort of 1,404 persons newly diagnosed with HIV in 2008–2009 followed for 24 months after linkage to care. Multivariable regression examined associations between individual (age, sex, race/ethnicity, HIV transmission risk, and insurance status) and community (economic deprivation, distance to care, access to public transit, and access to pharmacy services) factors and the outcomes: residence in a hotspot associated with poor retention in care and residence in a hotspot associated with poor viral suppression. Results 24.4% and 13.7% of persons resided in hotspots associated with poor retention and poor viral suppression, respectively. For persons residing in poor retention hotspots, 28.3% were retained in care compared to 40.4% of those residing outside hotspots (p<0.05). Similarly, for persons residing in poor viral suppression hotspots, 51.4% achieved viral suppression compared to 75.3% of those outside hotspots (p<.0.05). Factors significantly associated with residence in a poor retention hotspots included: female sex, lower economic deprivation, greater access to public transit, shorter distance to medical care, and longer distance to pharmacies. Factors significantly associated with residence in a poor viral suppression hotspots included; female sex, higher economic deprivation, and shorter distance to pharmacies. Conclusions Individual and community-level associations with geographic hotspots may inform both content and delivery strategies for interventions designed to improve retention in care and viral suppression. PMID:25867777

Individual and community factors associated with geographic clusters of poor HIV care retention and poor viral suppression.

PubMed

Eberhart, Michael G; Yehia, Baligh R; Hillier, Amy; Voytek, Chelsea D; Fiore, Danielle J; Blank, Michael; Frank, Ian; Metzger, David S; Brady, Kathleen A

2015-05-01

Previous analyses identified specific geographic areas in Philadelphia (hotspots) associated with negative outcomes along the HIV care continuum. We examined individual and community factors associated with residing in these hotspots. Retrospective cohort of 1404 persons newly diagnosed with HIV in 2008-2009 followed for 24 months after linkage to care. Multivariable regression examined associations between individual (age, sex, race/ethnicity, HIV transmission risk, and insurance status) and community (economic deprivation, distance to care, access to public transit, and access to pharmacy services) factors and the outcomes: residence in a hotspot associated with poor retention-in-care and residence in a hotspot associated with poor viral suppression. In total, 24.4% and 13.7% of persons resided in hotspots associated with poor retention and poor viral suppression, respectively. For persons residing in poor retention hotspots, 28.3% were retained in care compared with 40.4% of those residing outside hotspots (P < 0.05). Similarly, for persons residing in poor viral suppression hotspots, 51.4% achieved viral suppression compared with 75.3% of those outside hotspots (P < 0.0.05). Factors significantly associated with residence in poor retention hotspots included female sex, lower economic deprivation, greater access to public transit, shorter distance to medical care, and longer distance to pharmacies. Factors significantly associated with residence in poor viral suppression hotspots included female sex, higher economic deprivation, and shorter distance to pharmacies. Individual and community-level associations with geographic hotspots may inform both content and delivery strategies for interventions designed to improve retention-in-care and viral suppression.

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

PubMed Central

Hüser, Daniela; Weger, Stefan; Heilbronn, Regine

2003-01-01

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought. PMID:12663794

Assessing risk profiles for Salmonella serotypes in breeding pig operations in Portugal using a Bayesian hierarchical model

PubMed Central

2012-01-01

Background The EU Regulation No 2160/2003 imposes a reduction in the prevalence of Salmonella in pigs. The efficiency of control programmes for Salmonella in pigs, reported among the EU Member States, varies and definitive eradication seems very difficult. Control measures currently recommended for Salmonella are not serotype-specific. Is it possible that the risk factors for different Salmonella serotypes are different? The aim of this study was to investigate potential risk factors for two groups of Salmonella sp serotypes using pen faecal samples from breeding pig holdings representative of the Portuguese pig sector. Methods The data used come from the Baseline Survey for the Prevalence of Salmonella in breeding pigs in Portugal. A total of 1670 pen faecal samples from 167 herds were tested, and 170 samples were positive for Salmonella. The presence of Salmonella in each sample (outcome variable) was classified in three categories: i) no Salmonella, ii) Salmonella Typhimurium or S. Typhimurium-like strains with the antigenic formula: 1,4,5,12:i:-, , and iii) other serotypes. Along with the sample collection, a questionnaire concerning herd management and potential risk factors was utilised. The data have a “natural” hierarchical structure so a categorical multilevel analysis of the dataset was carried out using a Bayesian hierarchical model. The model was estimated using Markov Chain Monte Carlo methods, implemented in the software WinBUGS. Results The significant associations found (when compared to category “no Salmonella”), for category “serotype Typhimurium or S. Typhimurium-like strains with the antigenic formula: 1,4,5,12:i:-” were: age of breeding sows, size of the herd, number of pigs/pen and source of semen. For the category “other serotypes” the significant associations found were: control of rodents, region of the country, source of semen, breeding sector room and source of feed. Conclusions The risk factors significantly associated

Transmission probabilities and durations of immunity for three pathogenic group B Streptococcus serotypes

PubMed Central

Percha, Bethany; Newman, M. E. J.; Foxman, Betsy

2012-01-01

Group B Streptococcus (GBS) remains a major cause of neonatal sepsis and is an emerging cause of invasive bacterial infections. The 9 known serotypes vary in virulence, and there is little cross-immunity. Key parameters for planning an effective vaccination strategy, such as average length of immunity and transmission probabilities by serotype, are unknown. We simulated GBS spread in a population using a computational model with parameters derived from studies of GBS sexual transmission in a college dormitory. Here we provide estimates of the duration of immunity relative to the transmission probabilities for the 3 GBS serotypes most associated with invasive disease: Ia, III, and V. We also place upper limits on the durations of immunity for serotype Ia (570 days), III (1125 days) and V (260 days). Better transmission estimates are required to establish the epidemiological parameters of GBS infection and determine the best vaccination strategies to prevent GBS disease. PMID:21605704

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

PubMed Central

Schley, David; Whittaker, Robert J.; Neuman, Benjamin W.

2013-01-01

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations. PMID:23864502

Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds.

PubMed

Reeves, Andrew B; Poulson, Rebecca L; Muzyka, Denys; Ogawa, Haruko; Imai, Kunitoshi; Bui, Vuong Nghia; Hall, Jeffrey S; Pantin-Jackwood, Mary; Stallknecht, David E; Ramey, Andrew M

2016-06-01

Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we sequenced the fusion gene of 58 APMV-4 isolates collected in the United States, Japan and the Ukraine and compared them to all available sequences on GenBank. With only a single exception the phylogenetic clades of APMV-4 sequences were monophyletic with respect to their continents of origin (North America, Asia and Europe). Thus, we detected limited evidence for recent intercontinental dispersal of APMV-4 in this study. Published by Elsevier B.V.

Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds

PubMed Central

Reeves, Andrew B.; Poulson, Rebecca L.; Muzyka, Denys; Ogawa, Haruko; Imai, Kunitoshi; Bui, Vuong Nghia; Hall, Jeffrey S.; Pantin-Jackwood, Mary; Stallknecht, David E.; Ramey, Andrew M.

2016-01-01

Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we sequenced the fusion gene of 58 APMV-4 isolates collected in the United States, Japan and the Ukraine and compared them to all available sequences on GenBank. With only a single exception the phylogenetic clades of APMV-4 sequences were monophyletic with respect their continents of origin (North America, Asia and Europe). Thus, we detected limited evidence for recent intercontinental dispersal of APMV-4 in this study. PMID:26925702

Limited evidence of intercontinental dispersal of avian paramyxovirus serotype 4 by migratory birds

USGS Publications Warehouse

Reeves, Andrew; Poulson, Rebecca L.; Muzyka, Denys; Ogawa, Haruko; Imai, Kunitoshi; Nghia Bui, Vuong; Hall, Jeffrey S.; Pantin-Jackwood, Mary; Stallknecht, David E.; Ramey, Andrew M.

2016-01-01

Avian paramyxovirus serotype 4 (APMV-4) is a single stranded RNA virus that has most often been isolated from waterfowl. Limited information has been reported regarding the prevalence, pathogenicity, and genetic diversity of AMPV-4. To assess the intercontinental dispersal of this viral agent, we sequenced the fusion gene of 58 APMV-4 isolates collected in the United States, Japan and the Ukraine and compared them to all available sequences on GenBank. With only a single exception the phylogenetic clades of APMV-4 sequences were monophyletic with respect to their continents of origin (North America, Asia and Europe). Thus, we detected limited evidence for recent intercontinental dispersal of APMV-4 in this study.

Definition of human rotavirus serotypes by plaque reduction assay.

PubMed Central

Wyatt, R G; Greenberg, H B; James, W D; Pittman, A L; Kalica, A R; Flores, J; Chanock, R M; Kapikian, A Z

1982-01-01

Twenty different human rotavirus reassortants were characterized serologically by a plaque reduction assay as belonging to one of three distinct serotypes. Fourteen were similar if not identical to our prototype Wa strain; two were like the prototype DS-1 strain, and four belonged to a third serotype for which a prototype has not yet been selected. Hyperimmune sera raised against the three serotypes were required to distinguish among them, since postinfection sera had lower titers and were more cross-reactive than hyperimmune sera. These results confirmed the ability of a qualitative cytopathic neutralization test to predict correctly the Wa or DS-1 serotype. A strain of rhesus rotavirus (MMU 18006) was identified as belonging to the newly defined third serotype. Finally, an attempt was made to correlate previously published serotype analysis by neutralization of fluorescent cell-forming units with the results determined by the plaque reduction neutralization assay. PMID:6286487

Leucopenia associated with abalone viral ganglioneuritis.

PubMed

Hooper, C; Slocombe, R; Day, R; Crawford, S

2012-01-01

To compare microscopic lesion severity with circulating total haemocyte counts (THC) in abalone affected by abalone viral ganglioneuritis (AVG). A herpes-like virus led to severe mortality in a number of Australian abalone farms in 2006. The infection was associated with severe necrotising ganglioneuritis. The microscopic lesions were well demarcated, affecting the neural tissue almost exclusively and were characterised by necrosis and increased cellularity in affected ganglia and nerves. On two farms, the presence or absence of typical AVG pathology was compared with THC. Those abalone with microscopic lesions of AVG had significantly lower haemocyte counts. The mean THC in abalone with no evidence of AVG from both farms was 4.6 × 10(6)/mL (±0.3 SE). The THC in AVG-affected abalone in farm 1 was 2.8 × 10(6)/mL (±0.5 SE) and farm 2 was 0.98 × 10(6)/mL (±0.4 SE). Severe AVG is associated with leucopenia in affected abalone. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle.

PubMed

Anderson, Jenna; Hägglund, Sara; Bréard, Emmanuel; Riou, Mickaël; Zohari, Siamak; Comtet, Loic; Olofson, Ann-Sophie; Gélineau, Robert; Martin, Guillaume; Elvander, Marianne; Blomqvist, Gunilla; Zientara, Stéphan; Valarcher, Jean Francois

2014-11-20

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)-matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Role of dynamic capsomere supply for viral capsid self-assembly

NASA Astrophysics Data System (ADS)

Boettcher, Marvin A.; Klein, Heinrich C. R.; Schwarz, Ulrich S.

2015-02-01

Many viruses rely on the self-assembly of their capsids to protect and transport their genomic material. For many viral systems, in particular for human viruses like hepatitis B, adeno or human immunodeficiency virus, that lead to persistent infections, capsomeres are continuously produced in the cytoplasm of the host cell while completed capsids exit the cell for a new round of infection. Here we use coarse-grained Brownian dynamics simulations of a generic patchy particle model to elucidate the role of the dynamic supply of capsomeres for the reversible self-assembly of empty T1 icosahedral virus capsids. We find that for high rates of capsomere influx only a narrow range of bond strengths exists for which a steady state of continuous capsid production is possible. For bond strengths smaller and larger than this optimal value, the reaction volume becomes crowded by small and large intermediates, respectively. For lower rates of capsomere influx a broader range of bond strengths exists for which a steady state of continuous capsid production is established, although now the production rate of capsids is smaller. Thus our simulations suggest that the importance of an optimal bond strength for viral capsid assembly typical for in vitro conditions can be reduced by the dynamic influx of capsomeres in a cellular environment.

[Campylobacter jejuni O:19 serotype in Argentine poultry meat supply chain].

PubMed

Rossler, Eugenia; Fuhr, Estefanía M; Lorenzón, Guillermina; Romero-Scharpen, Analía; Berisvil, Ayelén P; Blajman, Jesica E; Astesana, Diego M; Zimmermann, Jorge A; Fusari, Marcia L; Signorini, Marcelo L; Soto, Lorena P; Frizzo, Laureano S; Zbrun, María V

Thermotolerant species of Campylobacter have been focus of attention in the last years because they are the major agent causing zoonotic foodborne diseases. In addition, Campylobacter jejuni O:19 serotype was associated with Guillain Barré syndrome. The aim of this study was to determine the proportion of C. jejuni O:19 serotype isolated at different stages of three poultry meat supply chain in Santa Fe, Argentina. The analysis showed that 18% of isolated C. jejuni belong to serotype O:19. It was also determined that the presence of these strains is given in almost all production stages. These results reflect a significant risk to public health of consumers. Epidemiological studies of Campylobacter should be considered to establish a risk manager policy. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

PubMed

Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

2018-03-16

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Intraspinal AAV Injections Immediately Rostral to a Thoracic Spinal Cord Injury Site Efficiently Transduces Neurons in Spinal Cord and Brain

PubMed Central

Klaw, Michelle C; Xu, Chen; Tom, Veronica J

2013-01-01

In the vast majority of studies utilizing adeno-associated virus (AAV) in central nervous system applications, including those published with spinal cord injury (SCI) models, AAV has been administered at the level of the cell body of neurons targeted for genetic modification, resulting in transduction of neurons in the vicinity of the injection site. However, as SCI interrupts many axon tracts, it may be more beneficial to transduce a diverse pool of supraspinal neurons. We determined if descending axons severed by SCI are capable of retrogradely transporting AAV to remotely transduce a variety of brain regions. Different AAV serotypes encoding the reporter green fluorescent protein (GFP) were injected into gray and white matter immediately rostral to a spinal transection site. This resulted in the transduction of thousands of neurons within the spinal cord and in multiple regions within the brainstem that project to spinal cord. In addition, we established that different serotypes had disparate regional specificity and that AAV5 transduced the most brain and spinal cord neurons. This is the first demonstration that retrograde transport of AAV by axons severed by SCI is an effective means to transduce a collection of supraspinal neurons. Thus, we identify a novel, minimally invasive means to transduce a variety of neuronal populations within both the spinal cord and the brain following SCI. This paradigm to broadly distribute viral vectors has the potential to be an important component of a combinatorial strategy to promote functional axonal regeneration. PMID:23881451

Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

PubMed

Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

2016-06-01

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

Dual Infection with Bluetongue Virus Serotypes and First-Time Isolation of Serotype 5 in India.

PubMed

Hemadri, D; Maan, S; Chanda, M M; Rao, P P; Putty, K; Krishnajyothi, Y; Reddy, G H; Kumar, V; Batra, K; Reddy, Y V; Maan, N S; Reddy, Y N; Singh, K P; Shivachandra, S B; Hegde, N R; Rahman, H; Mertens, P P C

2017-12-01

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks. © 2016 Blackwell Verlag GmbH.

Pasteurella multocida serotype 1 isolated from a lesser snow goose

USGS Publications Warehouse

Samuel, M.D.; Goldberg, Diana R.; Shadduck, D.J.; Price, J.I.; Cooch, E.G.

1997-01-01

Pharyngeal swabs were collected from 298 lesser snow geese (Chen caerulescens caerulescens) at Banks Island (Northwest Territories. Canada) in the summer of 1994. Pasteurella multocida serotype 1 was isolated from an adult male bird and P. multocida serotype 3 was isolated from an adult female goose. Pathogenicity of the serotype 1 isolate was confirmed by inoculation in Pekin ducks (Anas platyrhynchos). The serotype 3 isolate was non-pathogenic in Pekin ducks. This is the first documented isolation of pathogenic P. multocida serotype 1 from apparently healthy wild snow geese.

Factors Associated with Retention and Viral Suppression Among a Cohort of HIV+ Women of Color

PubMed Central

Fletcher, Jason; Verdecias, Niko; Garcia, Iliana; Blackstock, Oni; Cunningham, Chinazo

2015-01-01

Abstract Access to sustained HIV medical care is critical to achieving viral suppression. However, a variety of factors may impede or facilitate retention in care or becoming virally suppressed. Though retention and suppression are often treated separately, this study examined both in a cohort of 921 HIV+ women of color who participated in eight demonstration programs across the US. For women who met the inclusion criteria, 83% (n=587) were retained and 73% (n=357) were virally suppressed. Average age of women retained was 40.9, and 41.9 for those virally suppressed. The majority were African American/Black or Hispanic/Latina, single, and had no children less than 18 years of age, had health insurance, a high school degree or higher, were stably housed, and unemployed. Some factors associated with retention in care were indecision about seeking HIV medical care (AOR=0.42) and having children under the age of 18 (AOR=0.59). Some factors associated with being virally suppressed were living with others (AOR=0.58), current substance abuse (AOR=0.38), and fair/poor health (AOR=0.40). The findings suggest different processes and social mechanisms may influence retention and viral suppression. Interventions seeking to improve retention in care may require tailored program components and strategies that focus on improving viral suppression. PMID:25458205

Investigation of viral vectors using atomic force microscopy and microfluidic devices

NASA Astrophysics Data System (ADS)

Negishi, Atsuko

Researchers are modifying viruses into gene delivery vehicles in hope to cure diseases such as muscular dystrophy, hemophilia and cancer. Significant progress has been made toward this end, but further development and success of viral vectors depend on a deeper understanding of viral structure and physiology. Recent advances in microscopy have allowed new approaches to studying viruses that complement existing methodologies. Presented in this dissertation are novel viral studies using the atomic force microscope (AFM), a microscope that provides topographic information at the nanometer scale. As well microfluidic channels were used to study the effect of fluid flow properties on infection. A number of viruses are currently under study as potential vectors. We focus our studies on the adenovirus (Ad) and the adeno-associated virus (AAV) which have numerous attractive properties as vectors. The AFM is used to probe first, the structural aspects of the Ad and second, the virus-receptor interactions between AAV and its cell surface receptor, heparan sulfate proteoglycan (HSPG). The AFM was capable of imaging the capsid facets of intact Ad and DNA strands released from disrupted Ad capsids. In addition, we found that the stability of the capsid depended on the surface chemistry. An AFM-based binding assay was developed to study the binding between AAV and HSPG. The advantage of using the AFM for this purpose is its ability to simultaneously provide structural and quantitative information at the single molecule level. We measured a binding constant of 3.4 +/- 0.3 nM which is consistent with published reports. Microfluidic devices were used to study the dependence of fluid flow on infection. Cells were cultured in microfluidic channels and exposed to AAV vectors at various shear stresses. We found that a lower percentage of the cells were infected at higher shear stress. We also found that fluid forces can indirectly play a role in viral infection by influencing the cell

Serotype Distribution and Drug Resistance in Streptococcus pneumoniae, Palestinian Territories

PubMed Central

Kattan, Randa; Abu Rayyan, Amal; Zheiman, Inas; Idkeidek, Suzan; Baraghithi, Sabri; Rishmawi, Nabeel; Turkuman, Sultan; Abu-Diab, Afaf; Ghneim, Riyad; Zoughbi, Madeleine; Dauodi, Rula; Ghneim, Raed; Issa, Abed-El-Razeq; Siryani, Issa; Al Qas, Randa; Liddawi, Rawan; Khamash, Hatem; Kanaan, Moein; Marzouqa, Hiyam

2011-01-01

To determine antimicrobial drug resistance of Streptococcus pneumoniae serotypes, we analyzed isolates from blood cultures of sick children residing in the West Bank before initiation of pneumococcal vaccination. Of 120 serotypes isolated, 50.8%, 73.3%, and 80.8% of the bacteremia cases could have been prevented by pneumococcal conjugate vaccines. Serotype 14 was the most drug-resistant serotype isolated. PMID:21192863

High density recombinant AAV particles are competent vectors for in vivo transduction

USDA-ARS?s Scientific Manuscript database

Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

Streptococcus suis Bacterin and Subunit Vaccine Immunogenicities and Protective Efficacies against Serotypes 2 and 9▿†

PubMed Central

Baums, Christoph Georg; Kock, Christoph; Beineke, Andreas; Bennecke, Katharina; Goethe, Ralph; Schröder, Charlotte; Waldmann, Karl-Heinz; Valentin-Weigand, Peter

2009-01-01

Streptococcus suis causes numerous diseases in pigs, most importantly, meningitis, arthritis, septicemia, and bronchopneumonia. One of the major problems in modern swine production is the lack of a vaccine protecting against more than one S. suis serotype. The objective of this study was to determine the protective efficacy of a serotype 2 murein-associated protein (MAP) fraction subunit vaccine in comparison to that of a bacterin against experimental challenge with serotype 2 (containing muramidase-released protein [MRP], extracellular factor, and suilysin [SLY]) and serotype 9 (containing MRP variant MRP* and SLY) strains. MAP was shown to include different surface-associated proteins, such as the MRP and surface antigen one (SAO) expressed by both pathotypes used for challenge. The results of this study demonstrated that the serotype 2 bacterin induced protective immunity against homologous challenge. In contrast, the protective efficacy of the MAP subunit vaccine was low, though MAP immunization resulted in high serum immunoglobulin G2 titers against MRP and SAO. Importantly, immunization with bacterin but not with MAP induced opsonizing antibody titers against the serotype 2 strain, and these antibody titers were found to correlate with protection. However, after absorption with a nonencapsulated isogenic mutant, the sera from bacterin-immunized piglets failed to facilitate neutrophil killing, indicating that antibodies directed against capsule may not have been essential for opsonophagocytosis. Furthermore, induction of opsonizing antibodies against serotype 9 was not detectable in the group receiving bacterin or in the group receiving the MAP vaccine. In agreement, protection against the heterologous serotype 9 strain was low in both groups. Thus, identification of an antigen protecting against these two important S. suis pathotypes remains an important goal of future studies. PMID:19109449

Dengue virus life cycle: viral and host factors modulating infectivity.

PubMed

Rodenhuis-Zybert, Izabela A; Wilschut, Jan; Smit, Jolanda M

2010-08-01

Dengue virus (DENV 1-4) represents a major emerging arthropod-borne pathogen. All four DENV serotypes are prevalent in the (sub) tropical regions of the world and infect 50-100 million individuals annually. Whereas the majority of DENV infections proceed asymptomatically or result in self-limited dengue fever, an increasing number of patients present more severe manifestations, such as dengue hemorrhagic fever and dengue shock syndrome. In this review we will give an overview of the infectious life cycle of DENV and will discuss the viral and host factors that are important in controlling DENV infection.

Botulinum neurotoxin serotypes detected by electrochemical impedance spectroscopy.

PubMed

Savage, Alison C; Buckley, Nicholas; Halliwell, Jennifer; Gwenin, Christopher

2015-05-06

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A-E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins.

Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

PubMed Central

Savage, Alison C.; Buckley, Nicholas; Halliwell, Jennifer; Gwenin, Christopher

2015-01-01

Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins. PMID:25954998

Sensitivity and specificity of the AdenoPlus point-of-care system in detecting adenovirus in conjunctivitis patients at an ophthalmic emergency department: a diagnostic accuracy study.

PubMed

Kam, K Y Ronald; Ong, Hon Shing; Bunce, Catey; Ogunbowale, Lola; Verma, Seema

2015-09-01

To estimate the diagnostic accuracy (sensitivity and specificity) of the AdenoPlus point-of-care adenoviral test compared to PCR in an ophthalmic accident and emergency service. These findings were compared with those of a previous study. This was a prospective diagnostic accuracy study on 121 patients presenting to an emergency eye unit with a clinical picture of acute adenoviral conjunctivitis. AdenoPlus testing was carried out on one eye of each patient and a PCR analysis was also performed on a swab taken from the same eye. AdenoPlus and PCR results were interpreted by masked personnel. Sensitivity and specificity for the AdenoPlus test were calculated using PCR results as the reference standard. 121 patients were enrolled and 109 met the inclusion criteria. 43 patients (39.4%) tested positive for adenovirus by PCR analysis. The sensitivity of the AdenoPlus swab in detecting adenovirus was 39.5% (17/43, 95% CI 26% to 54%) and specificity was 95.5% (63/66, 95% CI 87% to 98%) compared to PCR. The AdenoPlus test has a high specificity for diagnosing adenoviral conjunctivitis, but in this clinical setting, we could not reproduce the high sensitivity that has been previously published. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will

ENHANCED GENE DELIVERY IN PORCINE VASCULATURE TISSUE FOLLOWING INCORPORATION OF ADENO-ASSOCIATED VIRUS NANOPARTICLES INTO POROUS SILICON MICROPARTICLES

PubMed Central

McConnell, Kellie I.; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E.

2014-01-01

There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells. PMID:25180449

Listeria monocytogenes Source Distribution Analysis Indicates Regional Heterogeneity and Ecological Niche Preference among Serotype 4b Clones.

PubMed

Lee, Sangmi; Chen, Yi; Gorski, Lisa; Ward, Todd J; Osborne, Jason; Kathariou, Sophia

2018-04-17

Biodiversity analysis of the foodborne pathogen Listeria monocytogenes recently revealed four serotype 4b major hypervirulent clonal complexes (CCs), i.e., CC1, CC2, CC4, and CC6. Hypervirulence was indicated by overrepresentation of these clones, and serotype 4b as a whole, among human clinical isolates in comparison to food. However, data on potential source-dependent partitioning among serotype 4b clones in diverse regions are sparse. We analyzed a panel of 347 serotype 4b isolates, primarily from North America, to determine the distribution of clones in humans, other animals, food, and water. CC1, CC2, CC4, and CC6 predominated, but surprisingly, only three clones, i.e., CC2 and the singleton sequence types (STs) ST382 and ST639, exhibited significant source-dependent associations, with higher propensity for food (CC2) or water (ST382 and ST639) than other sources. Pairwise comparisons between human and food isolates identified CC4 as the only serotype 4b clone significantly overrepresented among human isolates. Our analysis also revealed several serotype 4b clones emerging in North America. Two such emerging clones, ST382 (implicated in several outbreaks since 2014) and ST639, were primarily encountered among human and water isolates. Findings suggest that in spite of the ubiquity of CC1, CC2, CC4, and CC6, regional heterogeneity in serotype 4b is substantially larger than previously surmised. Analysis of even large strain panels from one region may not adequately predict clones unique to, and emerging in, other areas. Serotype 4b clonal complexes may differ in ecological niche preference, suggesting the need to further elucidate reservoirs and vehicles, especially for emerging clones. IMPORTANCE In Listeria monocytogenes , serotype 4b strains are leading contributors to human disease, but intraserotype distributions among different sources and regions remain poorly elucidated. Analysis of 347 serotype 4b isolates from four different sources, mostly from North

Serotyping, Genotyping, and Antimicrobial Susceptibility of Ornithobacterium rhinotracheale Isolates from Mexico.

PubMed

Peña-Vargas, Edgar Rafael; Vega-Sánchez, Vicente; Morales-Erasto, Vladimir; Trujillo-Ruíz, Héctor Hugo; Talavera-Rojas, Martín; Soriano-Vargas, Edgardo

2016-09-01

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease and septicemia in poultry. In this study, 9 reference strains and a total of 23 isolates of O. rhinotracheale from respiratory diseased poultry from Mexico were serotyped and genotyped. Furthermore, the antimicrobial susceptibility of isolates and reference strains of O. rhinotracheale were determined. All isolates belong to serotype A and showed a clonal relationship. All reference strains and isolates were resistant to colistin, fosfomycin, gentamicin, kanamycin, streptomycin, and trimethoprim-sulfamethoxazole. These results should eventually be helpful in planning strategies for the control of O. rhinotracheale infections in poultry in Mexico.

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing

PubMed Central

Davidsson, Marcus; Diaz-Fernandez, Paula; Schwich, Oliver D.; Torroba, Marcos; Wang, Gang; Björklund, Tomas

2016-01-01

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode. PMID:27874090

Group A Human Rotavirus Genomics: Evidence that Gene Constellations Are Influenced by Viral Protein Interactions▿ †

PubMed Central

Heiman, Erica M.; McDonald, Sarah M.; Barro, Mario; Taraporewala, Zenobia F.; Bar-Magen, Tamara; Patton, John T.

2008-01-01

Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication. PMID:18786998

The Assembly-Activating Protein Promotes Stability and Interactions between AAV's Viral Proteins to Nucleate Capsid Assembly.

PubMed

Maurer, Anna C; Pacouret, Simon; Cepeda Diaz, Ana Karla; Blake, Jessica; Andres-Mateos, Eva; Vandenberghe, Luk H

2018-05-08

The adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid's dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

PubMed

Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

2005-12-20

Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

Serotypes of Streptococcus suis isolated from healthy pigs in Phayao Province, Thailand.

PubMed

Thongkamkoon, P; Kiatyingangsulee, T; Gottschalk, M

2017-01-19

Streptococcus suis (S. suis) is an important swine and human pathogen. There are 33 serotypes that have been described. Zoonotic cases are very common the Northern part of Thailand, especially in Phayao Province. However, the prevalence of S. suis and, more particularly the different serotypes, in pigs in this region is poorly known and needed to be addressed. Distribution of S. suis serotypes varies depending on the geographical area. Knowledge of the serotype distribution is important for epidemiological studies. Consequently, 180 tonsil samples from slaughterhouse pigs in Phayao Province had been collected for surveillance, from which 196 S. suis isolates were recovered. Each isolate was subcultured and its serotype identified using multiplex PCR. Slide agglutination combined with precipitation tests were used following multiplex PCR to differentiate the isolates showing similar sizes of amplified products specific to either serotype 1 or 14 and 2 or 1/2. Non-typable isolates by multiplex PCR were serotyped by the coagglutination test. Of the 196 isolates, 123 (62.8%) were typable and 73 (37.2%) were non-typable. This study revealed the presence of serotypes 1, 1/2, 2, 3, 4, 5, 7, 9, 11, 12, 13, 14, 21, 22, 23, 24, 25, 29, and 30. Serotype 23 was the most prevalent (20/196, 10.2%), followed by serotype 9 (16/196, 8.2%), serotype 7 (16/196, 8.2%), and serotype 2 (11/196, 5.6%). The latter is the serotype responsible for most human cases. Almost all serotypes previously described are present in Northern Thailand. Therefore, this report provides useful data for future bacteriological studies.

Assignment of serotype to Salmonella enterica isolates obtained from poultry and their environment in southern Brazil

PubMed Central

Pulido-Landínez, M; Sánchez-Ingunza, R; Guard, J; do Nascimento, V Pinheiro

2013-01-01

To assess diversity of Salmonella enterica serotypes present in poultry and their environment from southern Brazil, the Kauffmann–White–Le Minor (KWL) scheme was used to serotype a total of 155 isolates. Isolates were then re-examined with nested PCR and sequencing of the dkgB-linked intergenic sequence ribotyping (ISR) region that assesses single nucleotide polymorphisms occurring around a 5S ribosomal gene. Serotypes identified were Heidelberg (40·6%), Enteritidis (34·2%), Hadar (8·4%), Typhimurium (3·9%), Gallinarum (3·2%), Agona (1·3%), Cerro (1·3%), Livingstone (1·3%), Infantis (0·6%), Isangi (0·6%), Mbandaka (0·6%), Montevideo (0·6%) and Senftenberg (0·6%). Three unique ISRs were detected from four strains. Day old chicks yielded only S. Enteritidis, whereas S. Heidelberg was most often associated with poultry carcasses. Overall agreement between KWL and ISR was 85·2%, with disagreement possibly due to the ability of ISR to detect mixtures of serotypes in culture. Overall, ISR provided more information than did KWL about the ecology of Salm. enterica on-farm. The O-antigen group D Salm. enterica serovars such as Pullorum, Gallinarum and Enteritidis appear susceptible to overgrowth by other serotypes. Significance and Impact of the Study Single nucleotide polymorphisms found in a group of poultry-associated Salmonella isolates from southern Brazil provided evidence of mixtures of serovar group D serotypes on-farm and in single samples from birds. This finding suggests that co-infection and interserotype competition of Salmonella enterica in poultry could impact the incidence of disease in animals or humans. In addition, unique serotypes were identified on-farm that escaped characterization by antibody typing. Application of cost-efficient and highly discriminatory genomic methods for assigning serotype may alter concepts about the epidemiology of Salm. enterica on-farm and in foods. PMID:23734786

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

PubMed

Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

1998-11-01

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products.

PubMed

Kim, Han Wool; Lee, Ji Hyen; Cho, Hye Kyung; Lee, Hyunju; Seo, Ho Seong; Lee, Soyoung; Kim, Kyung Hyo

2017-05-01

Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0-11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635-5,706 (serotype Ia), 488-1,421 (serotype Ib), and 962-3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups. © 2017 The Korean Academy of Medical Sciences.

Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products

PubMed Central

2017-01-01

Group B streptococcus (GBS) infection is a leading cause of sepsis and meningitis among infants, and is associated with high rates of morbidity and mortality in many countries. Protection against GBS typically involves antibody-mediated opsonization by phagocytes and complement components. The present study evaluated serotype-specific functional antibodies to GBS among Korean infants and in intravenous immunoglobulin (IVIG) products. An opsonophagocytic killing assay (OPA) was used to calculate the opsonization indices (OIs) of functional antibodies to serotypes Ia, Ib, and III in 19 IVIG products from 5 international manufacturers and among 98 Korean infants (age: 0–11 months). The GBS Ia, Ib, and III serotypes were selected because they are included in a trivalent GBS vaccine formulation that is being developed. The OI values for the IVIG products were 635–5,706 (serotype Ia), 488–1,421 (serotype Ib), and 962–3,315 (serotype III), and none of the IVIG lots exhibited undetectable OI values (< 4). The geometric mean OI values were similar for all 3 serotypes when we compared the Korean manufacturers. The seropositive rate among infants was significantly lower for serotype Ia (18.4%), compared to serotype Ib and serotype III (both, 38.8%). Infant age of ≥ 3 months was positively correlated with the seropositive rates for each serotype. Therefore, only a limited proportion of infants exhibited protective immunity against serotype Ia, Ib, and III GBS infections. IVIG products that exhibit high antibody titers may be a useful therapeutic or preventive measure for infants. Further studies are needed to evaluate additional serotypes and age groups. PMID:28378545

Intergenic Sequence Ribotyping using a region neighboring dkgB links genovar to Kauffman-White serotype of Salmonella enterica

USDA-ARS?s Scientific Manuscript database

Previous research identified that the 5S ribosomal (rrn) gene and associated flanking sequences that are closely linked to the dkgB gene of Salmonella enterica were highly variable between serotypes, but not between subpopulations within the same serotype (PMID: 17005008). The degree of variability ...

Lessons From Globally Coordinated Cessation of Serotype 2 Oral Poliovirus Vaccine for the Remaining Serotypes

PubMed Central

Duintjer Tebbens, Radboud J.

2017-01-01

Abstract Background. Comparing model expectations with the experience of oral poliovirus vaccine (OPV) containing serotype 2 (OPV2) cessation can inform risk management for the expected cessation of OPV containing serotypes 1 and 3 (OPV13). Methods. We compare the expected post-OPV2-cessation OPV2-related viruses from models with the evidence available approximately 6 months after OPV2 cessation. We also model the trade-offs of use vs nonuse of monovalent OPV (mOPV) for outbreak response considering all 3 serotypes. Results. Although too early to tell definitively, the observed die-out of OPV2-related viruses in populations that attained sufficiently intense trivalent OPV (tOPV) use prior to OPV2 cessation appears consistent with model expectations. As expected, populations that did not intensify tOPV use prior to OPV2 cessation show continued circulation of serotype 2 vaccine-derived polioviruses (VDPVs). Failure to aggressively use mOPV to respond to circulating VDPVs results in a high risk of uncontrolled outbreaks that would require restarting OPV. Conclusions. Ensuring a successful endgame requires more aggressive OPV cessation risk management than has occurred to date for OPV2 cessation. This includes maintaining high population immunity to transmission up until OPV13 cessation, meeting all prerequisites for OPV cessation, and ensuring sufficient vaccine supply to prevent and respond to outbreaks. PMID:28838198

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

Tuberculous meningitis is associated with higher cerebrospinal HIV-1 viral loads compared to other HIV-1-associated meningitides.

PubMed

Seipone, Ikanyeng D; Singh, Ravesh; Patel, Vinod B; Singh, Avashna; Gordon, Michelle L; Muema, Daniel M; Dheda, Keertan; Ndung'u, Thumbi

2018-01-01

To gain a better understanding of the immunopathogenesis of tuberculous meningitis (TBM) and identify potential diagnostic biomarkers that may discriminate TBM from other HIV-1-associated meningitides, we assessed HIV-1 viral load levels, drug resistance patterns in antiretroviral therapy (ART)-experienced patients with persistent viremia and soluble immunological analytes in peripheral blood and cerebrospinal fluid (CSF) of HIV-1 infected patients with TBM versus other meningitides. One hundred and three matched blood and CSF samples collected from HIV-1 infected patients with TBM or other meningitides presenting at a hospital in Durban, South Africa, from January 2009 to December 2011 were studied. HIV-1 RNA and 28 soluble immunological potential biomarkers were quantified in blood plasma and CSF. Viremic samples were assessed for HIV-1 drug resistance mutations. There were 16 TBM, 46 probable TBM, 35 non-TBM patients, and six unclassifiable patients. TBM and non-TBM patients did not differ in median plasma viral load but TBM patients had significantly higher median CSF viral load than non-TBM participants (p = 0.0005). No major drug resistance mutations were detected in viremic samples. Interleukin (IL)-1β, IL-17, platelet derived growth factor (PDGF)-BB, granulocyte colony stimulating factor (G-CSF) and cathelicidin were significantly elevated in the CNS of TBM participants compared to other patients although these associations were lost after correction for false discovery. Our data suggest that TB co-infection of the CNS is associated with enhanced localized HIV-1 viral replication but none of the evaluated soluble immunological potential biomarkers could reliably distinguish TBM from other HIV-associated meningitides.

Tuberculous meningitis is associated with higher cerebrospinal HIV-1 viral loads compared to other HIV-1-associated meningitides

PubMed Central

Seipone, Ikanyeng D.; Singh, Ravesh; Patel, Vinod B.; Singh, Avashna; Gordon, Michelle L.; Muema, Daniel M.; Dheda, Keertan

2018-01-01

To gain a better understanding of the immunopathogenesis of tuberculous meningitis (TBM) and identify potential diagnostic biomarkers that may discriminate TBM from other HIV-1-associated meningitides, we assessed HIV-1 viral load levels, drug resistance patterns in antiretroviral therapy (ART)-experienced patients with persistent viremia and soluble immunological analytes in peripheral blood and cerebrospinal fluid (CSF) of HIV-1 infected patients with TBM versus other meningitides. One hundred and three matched blood and CSF samples collected from HIV-1 infected patients with TBM or other meningitides presenting at a hospital in Durban, South Africa, from January 2009 to December 2011 were studied. HIV-1 RNA and 28 soluble immunological potential biomarkers were quantified in blood plasma and CSF. Viremic samples were assessed for HIV-1 drug resistance mutations. There were 16 TBM, 46 probable TBM, 35 non-TBM patients, and six unclassifiable patients. TBM and non-TBM patients did not differ in median plasma viral load but TBM patients had significantly higher median CSF viral load than non-TBM participants (p = 0.0005). No major drug resistance mutations were detected in viremic samples. Interleukin (IL)-1β, IL-17, platelet derived growth factor (PDGF)-BB, granulocyte colony stimulating factor (G-CSF) and cathelicidin were significantly elevated in the CNS of TBM participants compared to other patients although these associations were lost after correction for false discovery. Our data suggest that TB co-infection of the CNS is associated with enhanced localized HIV-1 viral replication but none of the evaluated soluble immunological potential biomarkers could reliably distinguish TBM from other HIV-associated meningitides. PMID:29394269

Temporal distribution of dengue virus serotypes in Colombian endemic area and dengue incidence. Re-introduction of dengue-3 associated to mild febrile illness and primary infection.

PubMed

Ocazionez, Raquel Elvira; Cortés, Fabián Mauricio; Villar, Luis Angel; Gómez, Sergio Yebrail

2006-11-01

We have investigated the temporal distribution of dengue (DEN) virus serotypes in the department (state) of Santander, Colombia, in relation to dengue incidence, infection pattern, and severity of disease. Viral isolation was attended on a total of 1452 acute serum samples collected each week from 1998 to 2004. The infection pattern was evaluated in 596 laboratory-positive dengue cases using an IgG ELISA, and PRNT test. The dengue incidence was documented by the local health authority. Predominance of DEN-1 in 1998 and DEN-3 re-introduction and predominance in 2001-2003 coincided with outbreaks. Predominance of DEN-2 in 2000-2001 coincided with more dengue hemorrhagic fever (DHF). DEN-4 was isolated in 2000-2001 and 2004 but was not predominant. There was an annual increase of primary dengue infections (from 13.7 to 81.4%) that correlated with frequency of DEN-3 (r = 0.83; P = 0.038). From the total number of primary dengue infections DEN-3 (81.3%) was the most frequent serotype. DHF was more frequent in DEN-2 infected patients than in DEN-3 infected patients: 27.5 vs 10.9% (P < 0.05). DEN-3 viruses belonged to subtype C (restriction site-specific-polymerase chain reaction) like viruses isolated in Sri-Lanka and other countries in the Americas. Our findings show the importance of continuous virological surveillance to identify the risk factors of dengue epidemics and severity.

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

PubMed Central

Webb, Stacy R.; Smith, Stacy E.; Fried, Michael G.

2018-01-01

ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

PubMed Central

Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

2017-01-01

ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the

Molecular diagnosis of non-serotypeable Shigella spp.: problems and prospects.

PubMed

Muthuirulandi Sethuvel, Dhiviya Prabaa; Devanga Ragupathi, Naveen Kumar; Anandan, Shalini; Walia, Kamini; Veeraraghavan, Balaji

2017-02-01

It is not always possible to identify Shigella serogroups/serotypes by biochemical properties alone. Specific identification requires serotyping. Occasionally, isolates that resemble Shigella spp. biochemically, but are non-agglutinable with available antisera, have been observed. Several mechanisms have been reported to limit the efficiency of the serotyping assay. Serotype conversion is a major mechanism in Shigella spp. to escape protective host immune responses. This easy conversion through significant modification of the O-antigen backbone results in different serotypes, which makes laboratory identification difficult. Furthermore, members of the family Enterobacteriaceae are closely related and there is antigenic cross-over (intra- and inter-specific cross-reaction) which affects the agglutination reaction. The performance of the available methods for identification of non-serotypeable Shigella is discussed here, and reveals them to be non-reliable. This shows a need for an alternative method for identification and typing of Shigella spp.

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

PubMed Central

Spencer, Brady L.; Shenoy, Anukul T.; Orihuela, Carlos J.

2017-01-01

ABSTRACT As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ. The coding sequence of wciZ contains eight consecutive TA repeats [(TA)8]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA)7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA)7 serotype 15C, and 15BΔwciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C. PMID:28637806

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies.

PubMed

Spencer, Brady L; Shenoy, Anukul T; Orihuela, Carlos J; Nahm, Moon H

2017-08-01

As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ The coding sequence of wciZ contains eight consecutive TA repeats [(TA) 8 ]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA) 7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA) 7 serotype 15C, and 15BΔ wciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C. Copyright © 2017 American Society for Microbiology.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

PubMed

Soto-Acosta, Rubén; Bautista-Carbajal, Patricia; Syed, Gulam H; Siddiqui, Aleem; Del Angel, Rosa M

2014-09-01

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong Li; Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL; Genetics Institute, University of Florida College of Medicine, Gainesville, FL

2008-11-25

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, theirmore » transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

Sustained viral gene delivery from a micro-fibrous, elastomeric cardiac patch to the ischemic rat heart.

PubMed

Gu, Xinzhu; Matsumura, Yasumoto; Tang, Ying; Roy, Souvik; Hoff, Richard; Wang, Bing; Wagner, William R

2017-07-01

Biodegradable and elastomeric patches have been applied to the surface of infarcted hearts as temporary mechanical supports to effectively alter adverse left ventricular remodeling processes. In this report, recombinant adeno-associated virus (AAV), known for its persistent transgene expression and low pathogenicity, was incorporated into elastomeric polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU) and processed by electrospinning into two formats (solid fibers and core-sheath fibers) designed to influence the controlled release behavior. The extended release of AAV encoding green fluorescent protein (GFP) was assessed in vitro. Sustained and localized viral particle delivery was achieved over 2 months in vitro. The biodegradable cardiac patches with or without AAV-GFP were implanted over rat left ventricular lesions three days following myocardial infarction to evaluate the transduction effect of released viral vectors. AAV particles were directly injected into the infarcted hearts as a control. Cardiac function and remodeling were significantly improved for 12 weeks after patch implantation compared to AAV injection. More GFP genes was expressed in the AAV patch group than AAV injection group, with both α-SMA positive cells and cardiac troponin T positive cells transduced in the patch group. Overall, the extended release behavior, prolonged transgene expression, and elastomeric mechanical properties make the AAV-loaded scaffold an attractive option for cardiac tissue engineering where both gene delivery and appropriate mechanical support are desired. Copyright © 2017. Published by Elsevier Ltd.

Slaughterhouse pigs are a major reservoir of Streptococcus suis serotype 2 capable of causing human infection in southern Vietnam.

PubMed

Ngo, Thi Hoa; Tran, Thi Bich Chieu; Tran, Thi Thu Nga; Nguyen, Van Dung; Campbell, James; Pham, Hong Anh; Huynh, Huu Tho; Nguyen, Van Vinh Chau; Bryant, Juliet E; Tran, Tinh Hien; Farrar, Jeremy; Schultsz, Constance

2011-03-28

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.

PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal.

PubMed

Bello Gonzalez, Teresita; Rivera-Olivero, Ismar Alejandra; Sisco, María Carolina; Spadola, Enza; Hermans, Peter W; de Waard, Jacobus H

2014-04-15

Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.

Update on: Shigella new serogroups/serotypes and their antimicrobial resistance.

PubMed

Muthuirulandi Sethuvel, D P; Devanga Ragupathi, N K; Anandan, S; Veeraraghavan, B

2017-01-01

Shigellosis represents a major burden of disease in developing countries. A low infectious dose allows the disease to be spread effectively. Although shigellosis is mostly a self-limiting disease, antibiotics are recommended to reduce deaths, disease symptoms and organism-shedding time. However, in India, antimicrobial resistance among the genus Shigella is more common than among any other enteric bacteria. Notably, new serotypes or subserotypes in Shigella are reported from various parts of the world. Identification of new subserotypes of Shigella spp. is becoming a major issue as these strains are nontypeable by conventional serotyping. The commercially available antisera may not cover all possible epitopes of the O lipopolysaccharide antigen of Shigella serotypes. Therefore, molecular methods which most closely approach the resolution of full serotyping are necessary to identify such strains. In addition, the knowledge of a prevalent serotype in various geographic regions may assist in formulating strategies such as the development of a vaccine to prevent infection especially when the immunity to disease is serotype specific, and to understand the disease burden caused by new Shigella serotypes. © 2016 The Society for Applied Microbiology.

Characterization of serological cross-reactivity between polysaccharide antigens of Streptococcus mutans serotypes c and d.

PubMed

Grossi, S; Prakobphol, A; Linzer, R; Campbell, L K; Knox, K W

1983-03-01

Immunological assays with antisera prepared against purified Streptococcus mutans serotype c polysaccharide demonstrated that a cross-reacting determinant on c polysaccharide reacted with the wall-associated rhamnose-glucose polysaccharide from S. mutans serotype d. Studies with 60 antisera prepared against chemostat cultures of S. mutans Ingbritt (c) demonstrated that the rhamnose-glucose polysaccharide cross-reactive determinant was consistently expressed on c antigen under a variety of growth conditions.

Viral Disease Networks?

NASA Astrophysics Data System (ADS)

Gulbahce, Natali; Yan, Han; Vidal, Marc; Barabasi, Albert-Laszlo

2010-03-01

Viral infections induce multiple perturbations that spread along the links of the biological networks of the host cells. Understanding the impact of these cascading perturbations requires an exhaustive knowledge of the cellular machinery as well as a systems biology approach that reveals how individual components of the cellular system function together. Here we describe an integrative method that provides a new approach to studying virus-human interactions and its correlations with diseases. Our method involves the combined utilization of protein - protein interactions, protein -- DNA interactions, metabolomics and gene - disease associations to build a ``viraldiseasome''. By solely using high-throughput data, we map well-known viral associated diseases and predict new candidate viral diseases. We use microarray data of virus-infected tissues and patient medical history data to further test the implications of the viral diseasome. We apply this method to Epstein-Barr virus and Human Papillomavirus and shed light into molecular development of viral diseases and disease pathways.

Comparison of oral toxicological properties of botulinum neurotoxin serotypes A and B.

PubMed

Cheng, Luisa W; Henderson, Thomas D

2011-07-01

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated proteins (NAPs), forming large complexes that have been shown to play important roles in oral toxicity. Using mouse intraperitoneal and oral models of botulism, we determined the dose response to both BoNT/B holotoxin and complex toxins, and compared the toxicities of BoNT/B and BoNT/A complexes. Although serotype A and B complexes have similar NAP composition, BoNT/B formed larger-sized complexes, and was approximately 90 times more lethal in mouse oral intoxications than BoNT/A complexes. When normalized by mean lethal dose, mice orally treated with high doses of BoNT/B complex showed a delayed time-to-death when compared with mice treated with BoNT/A complex. Furthermore, we determined the effect of various food matrices on oral toxicity of BoNT/A and BoNT/B complexes. BoNT/B complexes showed lower oral bioavailability in liquid egg matrices when compared to BoNT/A complexes. In summary, our studies revealed several factors that can either enhance or reduce the toxicity and oral bioavailability of BoNTs. Dissecting the complexities of the different BoNT serotypes and their roles in foodborne botulism will lead to a better understanding of toxin biology and aid future food risk assessments. Published by Elsevier Ltd.